Functional Peptide Analogs of PEDF

ABSTRACT

The invention provides compositions and methods relating to bioactive peptide analogs of PEDF.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 14/484,689, filed Sep. 12, 2014, which claims priority to U.S.Provisional Patent Application No. 61/877,335 filed Sep. 13, 2013 andU.S. Provisional Patent Application No. 61/951,854 filed Mar. 12, 2014,the contents of which are incorporated by reference herein in theirentirety.

BACKGROUND OF THE INVENTION

Pigment epithelium derived factor (PEDF) is a protein that is expressedin virtually all tissues of the human body, including nerve tissues(Tombran-Tink et al., 1995, J Neurosci. 15(7 Pt 1):4992-5003). PEDF isan angiogenesis inhibitor with neurotrophic properties and is a memberof the second group of the serpin family. (Tombran-Tink and Barnstable,2003, Nat Rev Neurosci. 4:628-636). PEDF has been shown to haveprotective effects which extend to neurons of the retina in particular,PEDF prevents damage to rat rod photoreceptors exposed to H₂O₂ orconstant bright light, to rods of the retinal degeneration (rd) andretinal degeneration slow (rds) mutant mice, and to Xenopus rods afterRPE detachment. (Cao et al., 1999, J Neurosci Res. 57(6):789-800;Cayouette et al., 1999, Neurobiol Dis. 6(6):523-532; Cao et al., 2001,Invest Opthalmol Vis Sci. 42(7):1646-1652; Jablonski et al., 2001, Glia35(1):14-25). Furthermore, reduced expression of PEDF plays a role ineye pathologies, including retinopathy and macular degeneration(Holekamp et al., 2002, Am J Opthalmol. 134:220-227). Increasedexpression of PEDF can induce regression of optical neovascularization(Mori et al., 2002, Invest Opthalmol V is Sci. 43:2428-34). PEDFexpression has also been shown to control tumor growth (Wang et al.,2003, Mol Ther. 2003; 8:72-79; Abe et al., 2004, Am J Pathol.164:1225-1232). Thus, PEDF has many therapeutic applications in avariety of pathologies involving angiogenesis and/or neuronaldegeneration.

Human PEDF is a 46,312 Da protein, which limits the effectiveness ofsome forms of delivery for therapeutic applications. The options foralternative delivery systems are limited for large polypeptides such asPEDF. One way in which this problem might be overcome is to identifyfragments of PEDF that maintain bioactivity. Identification offunctional fragments of PEDF is therefore desirable andstructure-function analysis on PEDF has been pursued (Becerra, 1997, AdvExp Med Biol. 425:223-37; Bilak et al., 2002, J Neurosci. 22:9378-9386;Filleur et al., 2005, Cancer Res. 65:5144-5251; Tombran-Tink et al.,2005, J Struc Biol. 151:130-150).

The neuroprotective and antiangiogenic activities of PEDF have beenlocalized to two short adjacent n-terminal fragments of the gene (Li etal., 2006, Exp. Eye. Res. 83:824-33; Gvritishvili et al., 2010, PLoSOne. 5:e15056; Bilak et al., 2002, J. Neurosci. 22:9378-86; Mirochnik etal., 2009, Clin. Cancer Res. 15:1655-63).

Given the various potential therapeutic uses of PEDF there is a need inthe art for molecules retaining the therapeutic activities offull-length PEDF but which are smaller in size and are easilyadministrable. The present invention addresses this need.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a composition comprisingat least one peptide analog of pigment epithelium derived factor (PEDF).In one embodiment, the at least one peptide analog comprises at leastone selected from the group consisting of SpxA1 (SEQ ID NO: 44), 81-1(SEQ ID NO: 1), 81-2 (SEQ ID NO: 2), 81-3 (SEQ ID NO: 3), 81-4 (SEQ IDNO: 4), 81-5 (SEQ ID NO: 5), 81-6 (SEQ ID NO: 6), 81-7 (SEQ ID NO: 7),81-8 (SEQ ID NO: 8), 81-9 (SEQ ID NO: 9), 81-10 (SEQ ID NO: 10), 81-11(SEQ ID NO: 11), 81-12 (SEQ ID NO: 12), 81-13 (SEQ ID NO: 12), 81-14(SEQ ID NO: 14), 81-15 (SEQ ID NO: 15), 81-16 (SEQ ID NO: 16), 81-17(SEQ ID NO: 17), 81-18 (SEQ ID NO: 18), 81-19 (SEQ ID NO: 19), and 81-20(SEQ ID NO: 20).

In one embodiment, the composition further comprises a pharmaceuticalcarrier. In one embodiment, the composition reduces at least one ofinflammation, the level of pro-inflammatory cytokines, cell death,vascular leakage, and macular edema.

In one embodiment, the at least one peptide analog comprises at leastone peptide analog selected from the group consisting of SpxA1 (SEQ IDNO: 44), 81-2 (SEQ ID NO: 2), 81-5 (SEQ ID NO: 5), 81-12 (SEQ ID NO:12), 81-13 (SEQ ID NO: 13), and 81-20 (SEQ ID NO: 20).

In one embodiment, the composition is selected from the group consistingof an eye drop, a gel, a foam, an injectate, and a cell. In oneembodiment, the composition is configured for delivery to the eye of asubject.

In one aspect the present invention provides a composition comprising atleast one isolated nucleic acid encoding at least one peptide analog ofPEDF or portion thereof. In one embodiment, the at least one peptideanalog comprises at least one selected from the group consisting ofSpxA1 (SEQ ID NO: 44), 81-1 (SEQ ID NO: 1), 81-2 (SEQ ID NO: 2), 81-3(SEQ ID NO: 3), 81-4 (SEQ ID NO: 4), 81-5 (SEQ ID NO: 5), 81-6 (SEQ IDNO: 6), 81-7 (SEQ ID NO: 7), 81-8 (SEQ ID NO: 8), 81-9 (SEQ ID NO: 9),81-10 (SEQ ID NO: 10), 81-11 (SEQ ID NO: 11), 81-12 (SEQ ID NO: 12),81-13 (SEQ ID NO: 12), 81-14 (SEQ ID NO: 14), 81-15 (SEQ ID NO: 15),81-16 (SEQ ID NO: 16), 81-17 (SEQ ID NO: 17), 81-18 (SEQ ID NO: 18),81-19 (SEQ ID NO: 19), and 81-20 (SEQ ID NO: 20).

In one embodiment, the at least one isolated nucleic acid comprises atleast one nucleotide sequence selected from the group consisting of SEQID NO: 45, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24,SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO:29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ IDNO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 41.

In one embodiment, the composition further comprises a pharmaceuticalcarrier. In one embodiment, the composition reduces at least one ofinflammation, the level of pro-inflammatory cytokines, cell death,vascular leakage, and macular edema.

In one embodiment, the at least one peptide analog comprises at leastone peptide analog selected from the group consisting of SpxA1 (SEQ IDNO: 44), 81-2 (SEQ ID NO: 2), 81-5 (SEQ ID NO: 5), 81-12 (SEQ ID NO:12), 81-13 (SEQ ID NO: 13), and 81-20 (SEQ ID NO: 20). In oneembodiment, the at least one isolated nucleic acid comprises at leastone nucleotide sequence selected from the group consisting of SEQ ID NO:45, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 32, SEQ ID NO: 33, and SEQID NO: 41.

In one embodiment, the composition is selected from the group consistingof an eye drop, a gel, a foam, a virus, an expression vector, aninjectate, and a cell. In one embodiment, the composition is configuredfor delivery to the eye of a subject.

In one aspect, the present invention provides a method for treating adisease or disorder in a subject, the method comprising administering toa subject a therapeutically effective amount of a composition comprisingat least one peptide analog of PEDF or isolated nucleic acid encodingthe same. In one embodiment, the at least one peptide analog comprisesat least one selected from the group consisting of SpxA1 (SEQ ID NO:44), 81-2 (SEQ ID NO: 2), 81-5 (SEQ ID NO: 5), 81-12 (SEQ ID NO: 12),81-13 (SEQ ID NO: 13) and 81-20 (SEQ ID NO: 20).

In one embodiment, the method reduces at least one of inflammation, thelevel of pro-inflammatory cytokines, cell death, vascular leakage, andmacular edema, in the subject.

In one embodiment, the composition is administered by a method selectedfrom the group consisting of administration as an eye drop,administration by intraocular injection, administration as a gel to theeye, administration as an implant in the eye that releases the peptideover time, administration as a virus that expresses the peptide, andadministration using a cell-based expression system.

In one embodiment, the disease or disorder is associated withangiogenesis. In one embodiment, the disease or disorder is selectedfrom the group consisting of diabetic retinopathy, retinopathy ofprematurity, age-related macular degeneration, retinitis pigmentosis,glaucoma, uveitis, corneal inflammation, diabetes, a neurodegenerativedisease, nerve injury, sepsis, acute respiratory distress syndrome,nephrotic syndrome, diabetic neuropathy, preproliferative diabeticretinopathy, proliferative diabetic retinopathy, cancer, and cysticfibrosis.

In one embodiment, the subject is a mammal. In one embodiment, themammal is a human.

In one aspect the present invention provides a method of reducing thelevel of VEGF in a subject, comprising administering to a subject acomposition comprising at least one peptide analog of PEDF or isolatednucleic acid encoding the same. In one embodiment, the at least onepeptide analog comprises at least one selected from the group consistingof 81-2 (SEQ ID NO: 2), 81-5 (SEQ ID NO: 5), 81-10 (SEQ ID NO: 10),81-12 (SEQ ID NO: 12), 81-13 (SEQ ID NO: 13), 81-19 (SEQ ID NO: 19) and81-20 (SEQ ID NO: 20).

In one aspect, the present invention provides a biological assaycomprising contacting a cell with a composition comprising at least onepeptide analog of PEDF, wherein the at least one peptide analogcomprises at least one selected from the group consisting of SpxA1 (SEQID NO: 44), 81-1 (SEQ ID NO: 1), 81-2 (SEQ ID NO: 2), 81-3 (SEQ ID NO:3), 81-4 (SEQ ID NO: 4), 81-5 (SEQ ID NO: 5), 81-6 (SEQ ID NO: 6), 81-7(SEQ ID NO: 7), 81-8 (SEQ ID NO: 8), 81-9 (SEQ ID NO: 9), 81-10 (SEQ IDNO: 10), 81-11 (SEQ ID NO: 11), 81-12 (SEQ ID NO: 12), 81-13 (SEQ ID NO:12), 81-14 (SEQ ID NO: 14), 81-15 (SEQ ID NO: 15), 81-16 (SEQ ID NO:16), 81-17 (SEQ ID NO: 17), 81-18 (SEQ ID NO: 18), 81-19 (SEQ ID NO:19), and 81-20 (SEQ ID NO: 20).

In one embodiment, the assay comprises contacting the cell with a liquidmedium comprising the at least one peptide analog. In one embodiment theassay comprises contacting a cell with an isolated nucleic acid encodingthe at least one peptide analog and wherein the cell secretes the atleast one peptide analog.

In one embodiment, the assay comprises detecting the effect of thecomposition on at least one selected from the group consisting ofinflammation, cell death, vascular leakage, tumor growth, apoptosis,cytoskeletal function, mitochondrial function, oxidative stress,angiogenesis, neurogenesis, cell growth, immune function, celldifferentiation, adipogenesis, bone deposition, and gene expression. Inone embodiment, the assay further comprises contacting the cell with atest compound.

In one aspect, the present invention provides a method of generating anantibody, comprising immunizing a subject with a composition comprisingat least one immunogenic peptide analog of PEDF or isolated nucleic acidencoding the same under conditions suitable for eliciting an immuneresponse, and recovering an antibody from the subject, wherein the atleast one immunogenic peptide analog comprises at least one selectedfrom the group consisting of SpxA1 (SEQ ID NO: 44), 81-1 (SEQ ID NO: 1),81-2 (SEQ ID NO: 2), 81-3 (SEQ ID NO: 3), 81-4 (SEQ ID NO: 4), 81-5 (SEQID NO: 5), 81-6 (SEQ ID NO: 6), 81-7 (SEQ ID NO: 7), 81-8 (SEQ ID NO:8), 81-9 (SEQ ID NO: 9), 81-10 (SEQ ID NO: 10), 81-11 (SEQ ID NO: 11),81-12 (SEQ ID NO: 12), 81-13 (SEQ ID NO: 12), 81-14 (SEQ ID NO: 14),81-15 (SEQ ID NO: 15), 81-16 (SEQ ID NO: 16), 81-17 (SEQ ID NO: 17),81-18 (SEQ ID NO: 18), 81-19 (SEQ ID NO: 19), and 81-20 (SEQ ID NO: 20).In one embodiment, the antibody binds to PEDF or a fragment thereof.

In one embodiment, the present invention provides an antibody thatspecifically binds to a peptide, wherein the peptide comprises at leastone of the group consisting of PEDF, a fragment of PEDF, and an analogof PEDF. In one embodiment, the peptide is a peptide analog selectedfrom the group consisting of SpxA1 (SEQ ID NO: 44), 81-1 (SEQ ID NO: 1),81-2 (SEQ ID NO: 2), 81-3 (SEQ ID NO: 3), 81-4 (SEQ ID NO: 4), 81-5 (SEQID NO: 5), 81-6 (SEQ ID NO: 6), 81-7 (SEQ ID NO: 7), 81-8 (SEQ ID NO:8), 81-9 (SEQ ID NO: 9), 81-10 (SEQ ID NO: 10), 81-11 (SEQ ID NO: 11),81-12 (SEQ ID NO: 12), 81-13 (SEQ ID NO: 12), 81-14 (SEQ ID NO: 14),81-15 (SEQ ID NO: 15), 81-16 (SEQ ID NO: 16), 81-17 (SEQ ID NO: 17),81-18 (SEQ ID NO: 18), 81-19 (SEQ ID NO: 19), and 81-20 (SEQ ID NO: 20).

In one embodiment, the present invention provides a method of culturinga stem cell comprising contacting the stem cell with at least onepeptide analog of PEDF. In one embodiment, the at least peptide analogcomprises at least one selected from the group consisting of SpxA1 (SEQID NO: 44), 81-1 (SEQ ID NO: 1), 81-2 (SEQ ID NO: 2), 81-3 (SEQ ID NO:3), 81-4 (SEQ ID NO: 4), 81-5 (SEQ ID NO: 5), 81-6 (SEQ ID NO: 6), 81-7(SEQ ID NO: 7), 81-8 (SEQ ID NO: 8), 81-9 (SEQ ID NO: 9), 81-10 (SEQ IDNO: 10), 81-11 (SEQ ID NO: 11), 81-12 (SEQ ID NO: 12), 81-13 (SEQ ID NO:12), 81-14 (SEQ ID NO: 14), 81-15 (SEQ ID NO: 15), 81-16 (SEQ ID NO:16), 81-17 (SEQ ID NO: 17), 81-18 (SEQ ID NO: 18), 81-19 (SEQ ID NO:19), and 81-20 (SEQ ID NO: 20).

In one embodiment, the method comprises contacting the cell with aliquid medium comprising the at least one peptide analog. In oneembodiment, the method comprises contacting a cell with an isolatednucleic acid encoding the at least one peptide analog and wherein thecell secretes the at least one peptide analog.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of illustrating the invention, there are depicted in thedrawings certain embodiments of the invention. However, the invention isnot limited to the precise arrangements and instrumentalities of theembodiments depicted in the drawings.

FIG. 1 is a schematic of PEDF, P78 and spxA1.

FIG. 2A and FIG. 2B are a series of graphs demonstrating the effects ofthe peptides to increase cell viability by increasing levels of ATP weretested in two models of cell death: (FIG. 2A) serum starvation; (FIG.2B) oxidative stress caused by hydrogen peroxide toxicity (300 μM).

FIG. 3A through FIG. 3C are a series of graphs depicting the results ofin vitro experiments in RPE cultures. The graphs demonstrate the effectsof each peptide on the inflammatory and vascularpermeability/angiogenesis cytokines TNFα (FIG. 3A), IFNγ (FIG. 3B), andVEGFA (FIG. 3C) compared to P78.

FIG. 4A and FIG. 4B are a series of images depicting the ability of thepeptides to reduce vascular leakage and stability of the peptides. Thepeptides resulted in increase in the tight junction proteins ZO1 andoccludin (FIG. 4A). The results demonstrate that the peptides were morestable compared to P78 and SpxA1 (FIG. 4B).

FIG. 5A through FIG. 5D are a series of graphs depicting the CD spectrafor various peptides, demonstrating the helical structure of thepeptides.

FIG. 6 is a graph depicting the peptide bioavailability of selectedpeptides in C57BL/6 mice vitreous.

FIG. 7 is a set of images depicting the presence of delivered peptideanalogs in the retina layers 1 hour following administrating of peptideeye drops in diabetic Ins2Akita mice.

FIG. 8 is a set of graphs demonstrating the reduction of proinflammatorycytokines (IFN-γ, TNF-α, and IL-6) in diabetic mice, mediated byadministration of selected peptides.

FIG. 9 is a graph demonstrating the reduction in VEGF levels in diabeticmice, mediated by administration of selected peptides.

FIG. 10 is a graph demonstrating the reduction in albumin leakage in theretina of diabetic mice, mediated by administration of selectedpeptides.

FIG. 11 is a set of images (obtained at 20× magnification) depictingretinal sections labeled for albumin, demonstrating that diabeticretinas contained higher levels of albumin throughout the retinalparenchyma and in blood vessels.

FIG. 12A through FIG. 12F is a set of images of retinal sections ofdiabetic mice labeled for albumin. FIG. 12A (40×) depicts increasedalbumin levels in the photoreceptor inner and outer segment areas andvascular leakage into the retinal parenchyma in the outer plexiformlayers. FIG. 12B (40×), FIG. 12C (40×), FIG. 12E (80×) and FIG. 12F(80×) depict vascular leakage in the retinal ganglion layer. FIG. 12D(40×) depicts albumin in a large blood vessel in the retinal ganglionlayer.

FIG. 13 is a set of images depicting the effects of the selectedpeptides on vascular leakage as measured by albumin extravasation intothe retina.

FIG. 14 is a graph depicting an increase in the survival of retinalganglion cells in peptide treated diabetic mice.

FIG. 15 is a set of images depicting DAPI and TUNEL stained retinas ofpeptide treated diabetic mice and controls.

FIG. 16A through FIG. 16C depict the results of experimentsinvestigating the activity of the PEDF-derived peptides on HUVEC tubeformation. Representative images of VEGF-mediated HUVEC tube formationare show (blue) on layers of human dermal fibroblast (grey). Effects ofVEGF alone (4 ng/mL), and Suramin (100 μM) are shown in FIG. 16A, whilethe effects of PEDF peptides P78, 81-2, 81-5, 81-12, 81-13, 81-20 (100nM) are shown in FIG. 16B. Results depict HUVEC tube formation over 12days. HUVEC CytoLight green (green) represent endothelial cells notforming tubes. The data in triplicate cultures is quantified in thegraphs of FIG. 16C, showing the average HUVEC network length over 12days.

FIG. 17 is set of graphs depicting the regulation of transcriptionfactors by PEDF-derived peptides. The demonstrates suggests that PEDFand the active peptide region (P78) or a truncated sequence of thisregion (Spx) regulates transcription factors (TFs) that controlexpression of a wide range of genes that are involved in varied andcomplementary processes. These include TFs regulating genotoxic stress,environmental stress, and apoptosis (HiNF, Nrf1), cell cycle progressionand cell differentiation (RB), and TFs controlling ER stress andinflammatory response (CCAAT/C-EBP).

FIG. 18A and FIG. 18B depict the results of experiments investigatingthe regulation of inflammatory cytokines by PEDF-derived peptides. Eachgraph depicts the regulation of a particular cytokine for the varioustreatments (from left to right—control, P78, SpxA1, 81-2, 81-5, 81-12,81-13, and 81-20).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides peptide analogs of PEDF and uses thereof.The invention is based in part on the discovery that peptides andpeptide analogs based on p′78, a 44 residue peptide from PEDF (residues78 to 121 see FIG. 1) retain various desirable therapeutic propertiesand modifications and/or changes to the natural sequence providespeptides with enhanced therapeutic benefits. Accordingly, the inventionprovides compositions comprising small functional peptide analogs ofPEDF and uses thereof.

In particular, the present invention is based on the discovery thatpeptide analogs based on the active p78 fragment of PEDF, includingcertain peptides having internal amino acids substitutions, hydrophobiccaps and modifications to support helix stabilization have desiredactivities of PEDF and in some cases have enhanced activity. In oneembodiment, peptides of the present invention are provided in Table 1and are referred to herein as SpxA1 and 81-1 to 81-20.

The invention provides compositions comprising peptide analogs and/orisolated nucleic acids encoding peptide analogs useful for a widevariety of applications. The invention also provides methods of usingthe compositions as well as kits containing the compositions. The smallpeptides of the invention allow relative ease in crossing tissuebarriers, can be synthesized in reproducibly pure large-scalequantities, and result in fewer side effects compared to full lengthPEDF. In certain embodiments the compositions of the invention can beprepared and administered as eye drops which provide major advantagesover prior art treatment methods.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Generally,the nomenclature used herein and the laboratory procedures in cellculture, molecular genetics, organic chemistry, and nucleic acidchemistry and hybridization are those well-known and commonly employedin the art.

Standard techniques are used for nucleic acid and peptide synthesis. Thetechniques and procedures are generally performed according toconventional methods in the art and various general references (e.g.,Sambrook and Russell, 2012, Molecular Cloning, A Laboratory Approach,Cold Spring Harbor Press, Cold Spring Harbor, N.Y., and Ausubel et al.,2005, Current Protocols in Molecular Biology, John Wiley & Sons, NY),which are provided throughout this document.

The nomenclature used herein and the laboratory procedures used inanalytical chemistry and organic syntheses described below are thosewell-known and commonly employed in the art. Standard techniques ormodifications thereof, are used for chemical syntheses and chemicalanalyses.

As used herein, amino acids are represented by the full name thereof, bythe three-letter code as well as the one-letter code correspondingthereto:

3-Letter 1-Letter Full Name Code Code Alanine Ala A Arginine Arg RAsparagine Asn N Aspartic Acid Asp D Cysteine Cys C Glutamic Acid Glu EGlutamine Gln Q Glycine Gly G Histidine His H Isoleucine Ile I LeucineLeu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro PSerine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine ValV

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

As used herein, the term “about” will be understood by persons ofordinary skill in the art and will vary to some extent on the context inwhich it is used. As used herein when referring to a measurable valuesuch as an amount, a temporal duration, and the like, the term “about”is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% fromthe specified value, as such variations are appropriate to perform thedisclosed methods.

The term “abnormal” when used in the context of organisms, tissues,cells or components thereof, refers to those organisms, tissues, cellsor components thereof that differ in at least one observable ordetectable characteristic (e.g., age, treatment, time of day, etc.) fromthose organisms, tissues, cells or components thereof that display the“normal” (expected) respective characteristic. Characteristics which arenormal or expected for one cell or tissue type, might be abnormal for adifferent cell or tissue type.

As used herein, a disease or disorder is “alleviated” if the severity ofa symptom of the disease or disorder, the frequency with which such asymptom is experienced by a patient, or both, are reduced.

As used herein, the terms “complementary” or “complementarity” are usedin reference to polynucleotides (i.e., a sequence of nucleotides)related by the base-pairing rules. For example, the sequence “A-G-T,” iscomplementary to the sequence “T-C-A.” Complementarity may be “partial,”in which only some of the nucleic acids' bases are matched according tothe base pairing rules. Or, there may be “complete” or “total”complementarity between the nucleic acids. The degree of complementaritybetween nucleic acid strands has significant effects on the efficiencyand strength of hybridization between nucleic acid strands. This is ofparticular importance in amplification reactions, as well as detectionmethods that depend upon binding between nucleic acids.

As used herein, the terms “conservative variation” or “conservativesubstitution” as used herein refers to the replacement of an amino acidresidue by another, biologically similar residue. Conservativevariations or substitutions are not likely to substantially change theshape and/or activity of the peptide chain. Families of amino acidresidues having side chains with similar charges have been defined inthe art. These families include amino acids with basic side chains(e.g., lysine, arginine, histidine), acidic side chains (e.g., asparticacid, glutamic acid), uncharged polar side chains (e.g., glycine,asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolarside chains (e.g., alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan), beta-branched side chains (e.g.,threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine).

A “disease” is a state of health of an animal wherein the animal cannotmaintain homeostasis, and wherein if the disease is not ameliorated thenthe animal's health continues to deteriorate. In contrast, a “disorder”in an animal is a state of health in which the animal is able tomaintain homeostasis, but in which the animal's state of health is lessfavorable than it would be in the absence of the disorder. Leftuntreated, a disorder does not necessarily cause a further decrease inthe animal's state of health.

An “effective amount” as used herein, means an amount which provides atherapeutic or prophylactic benefit.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

As used herein, the term “fragment,” as applied to a nucleic acid,refers to a subsequence of a larger nucleic acid. A “fragment” of anucleic acid can be at least about 15 nucleotides in length; forexample, at least about 50 nucleotides to about 100 nucleotides; atleast about 100 to about 500 nucleotides, at least about 500 to about1000 nucleotides; at least about 1000 nucleotides to about 1500nucleotides; about 1500 nucleotides to about 2500 nucleotides; or about2500 nucleotides (and any integer value in between). As used herein, theterm “fragment,” as applied to a protein or peptide, refers to asubsequence of a larger protein or peptide. A “fragment” of a protein orpeptide can be at least about 20 amino acids in length; for example, atleast about 50 amino acids in length; at least about 100 amino acids inlength; at least about 200 amino acids in length; at least about 300amino acids in length; or at least about 400 amino acids in length (andany integer value in between).

“Homologous” refers to the sequence similarity or sequence identitybetween two polypeptides or between two nucleic acid molecules. When aposition in both of the two compared sequences is occupied by the samebase or amino acid monomer subunit, e.g., if a position in each of twoDNA molecules is occupied by adenine, then the molecules are homologousat that position. The percent of homology between two sequences is afunction of the number of matching or homologous positions shared by thetwo sequences divided by the number of positions compared X 100. Forexample, if 6 of 10 of the positions in two sequences are matched orhomologous then the two sequences are 60% homologous. By way of example,the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, acomparison is made when two sequences are aligned to give maximumhomology.

“Instructional material,” as that term is used herein, includes apublication, a recording, a diagram, or any other medium of expressionwhich can be used to communicate the usefulness of the nucleic acid,peptide, and/or compound of the invention in the kit for identifying,diagnosing or alleviating or treating the various diseases or disordersrecited herein. Optionally, or alternately, the instructional materialmay describe one or more methods of identifying, diagnosing oralleviating the diseases or disorders in a cell or a tissue of asubject. The instructional material of the kit may, for example, beaffixed to a container that contains the nucleic acid, peptide, and/orcompound of the invention or be shipped together with a container thatcontains the nucleic acid, peptide, and/or compound. Alternatively, theinstructional material may be shipped separately from the container withthe intention that the recipient uses the instructional material and thecompound cooperatively.

“Isolated” means altered or removed from the natural state. For example,a nucleic acid or a peptide naturally present in a living animal is not“isolated,” but the same nucleic acid or peptide partially or completelyseparated from the coexisting materials of its natural state is“isolated.” An isolated nucleic acid or protein can exist insubstantially purified form, or can exist in a non-native environmentsuch as, for example, a host cell.

The term “label” when used herein refers to a detectable compound orcomposition that is conjugated directly or indirectly to a probe togenerate a “labeled” probe. The label may be detectable by itself (e.g.radioisotope labels or fluorescent labels) or, in the case of anenzymatic label, may catalyze chemical alteration of a substratecompound or composition that is detectable (e.g., avidin-biotin). Insome instances, primers can be labeled to detect a PCR product.

By the term “modulating,” as used herein, is meant mediating adetectable increase or decrease in the level of a mRNA, polypeptide, ora response in a subject compared with the level of a mRNA, polypeptideor a response in the subject in the absence of a treatment or compound,and/or compared with the level of a mRNA, polypeptide, or a response inan otherwise identical but untreated subject. The term encompassesperturbing and/or affecting a native signal or response therebymediating a beneficial therapeutic response in a subject, preferably, ahuman.

“Neurotrophic activity” as used herein is the ability to enhancesurvival and/or growth of neuronal cell populations. Neuroprotectiveactivity, such as delaying or reducing neuronal apoptosis, isencompassed by the term neurotrophic activity.

A “nucleic acid” refers to a polynucleotide and includespoly-ribonucleotides and poly-deoxyribonucleotides. Nucleic acidsaccording to the present invention may include any polymer or oligomerof pyrimidine and purine bases, preferably cytosine, thymine, anduracil, and adenine and guanine, respectively. (See Albert L. Lehninger,Principles of Biochemistry, at 793-800 (Worth Pub. 1982) which is hereinincorporated in its entirety for all purposes). Indeed, the presentinvention contemplates any deoxyribonucleotide, ribonucleotide orpeptide nucleic acid component, and any chemical variants thereof, suchas methylated, hydroxymethylated or glucosylated forms of these bases,and the like. The polymers or oligomers may be heterogeneous orhomogeneous in composition, and may be isolated from naturally occurringsources or may be artificially or synthetically produced. In addition,the nucleic acids may be DNA or RNA, or a mixture thereof, and may existpermanently or transitionally in single-stranded or double-strandedform, including homoduplex, heteroduplex, and hybrid states.

An “oligonucleotide” or “polynucleotide” is a nucleic acid ranging fromat least 2, preferably at least 8, 15 or 25 nucleotides in length, butmay be up to 50, 100, 1000, or 5000 nucleotides long or a compound thatspecifically hybridizes to a polynucleotide. Polynucleotides includesequences of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) ormimetics thereof which may be isolated from natural sources,recombinantly produced or artificially synthesized. A further example ofa polynucleotide of the present invention may be a peptide nucleic acid(PNA). (See U.S. Pat. No. 6,156,501 which is hereby incorporated byreference in its entirety.) The invention also encompasses situations inwhich there is a nontraditional base pairing such as Hoogsteen basepairing which has been identified in certain tRNA molecules andpostulated to exist in a triple helix. “Polynucleotide” and“oligonucleotide” are used interchangeably in this disclosure. It willbe understood that when a nucleotide sequence is represented herein by aDNA sequence (e.g., A, T, G, and C), this also includes thecorresponding RNA sequence (e.g., A, U, G, C) in which “U” replaces “T”.

By describing two peptides or polypeptides as “operably fused” is meantthat the structure and/or biological activity of each individual peptideis also present in the fusion.

The terms “patient,” “subject,” “individual,” and the like are usedinterchangeably herein, and refer to any animal, or cells thereofwhether in vitro or in situ, amenable to the methods described herein.In certain non-limiting embodiments, the patient, subject or individualis a human.

As used herein, the terms “peptide,” “polypeptide,” and “protein” areused interchangeably, and refer to a compound comprised of amino acidresidues covalently linked by peptide bonds. A protein or peptide mustcontain at least two amino acids, and no limitation is placed on themaximum number of amino acids that can comprise a protein's or peptide'ssequence. Polypeptides include any peptide or protein comprising two ormore amino acids joined to each other by peptide bonds. As used herein,the term refers to both short chains, which also commonly are referredto in the art as peptides, oligopeptides and oligomers, for example, andto longer chains, which generally are referred to in the art asproteins, of which there are many types. “Polypeptides” include, forexample, biologically active fragments, substantially homologouspolypeptides, oligopeptides, homodimers, heterodimers, variants ofpolypeptides, modified polypeptides, derivatives, analogs, fusionproteins, among others. The polypeptides include natural peptides,recombinant peptides, synthetic peptides, or a combination thereof.

As used herein, a “peptidomimetic” is a compound containing non-peptidicstructural elements that is capable of mimicking the biological actionof a parent peptide. A peptidomimetic may or may not comprise peptidebonds.

As used herein, “polynucleotide” includes cDNA, RNA, DNA/RNA hybrid,antisense RNA, ribozyme, genomic DNA, synthetic forms, and mixedpolymers, both sense and antisense strands, and may be chemically orbiochemically modified to contain non-natural or derivatized, synthetic,or semi-synthetic nucleotide bases. Also, contemplated are alterationsof a wild type or synthetic gene, including but not limited to deletion,insertion, substitution of one or more nucleotides, or fusion to otherpolynucleotide sequences.

A “prophylactic” treatment is a treatment administered to a subject whodoes not exhibit signs of a disease or exhibits only early signs of thedisease for the purpose of decreasing the risk of developing pathologyassociated with the disease.

By the term “retinopathy” as used herein, is meant the abnormaldevelopment of blood vessels within or around the retina that may or maynot enter the vitreous. Injury, disease, ischemic events, laser or otheriatrogenic treatments may induce retinopathy.

By the term “signal sequence” is meant a polynucleotide sequence whichencodes a peptide that directs the path a polypeptide takes within acell, i.e., it directs the cellular processing of a polypeptide in acell, including, but not limited to, eventual secretion of a polypeptidefrom a cell. A signal peptide is a sequence of amino acids which aretypically, but not exclusively, found at the amino terminus of apolypeptide which targets the synthesis of the polypeptide to theendoplasmic reticulum. In some instances, the signal peptide isproteolytically removed from the polypeptide and is thus absent from themature protein.

The term “substantially pure” describes a compound, e.g., a protein orpolypeptide, which has been separated from components which naturallyaccompany it. Typically, a compound is substantially pure when at least10%, more preferably at least 20%, more preferably at least 50%, morepreferably at least 60%, more preferably at least 75%, more preferablyat least 90%, and most preferably at least 99% of the total material (byvolume, by wet or dry weight, or by mole percent or mole fraction) in asample is the compound of interest. Purity can be measured by anyappropriate method, e.g., in the case of polypeptides, by columnchromatography, gel electrophoresis or HPLC analysis. A compound, e.g.,a protein, is also substantially purified when it is essentially free ofnaturally associated components or when it is separated from the nativecontaminants which accompany it in its natural state.

As used herein, the term “substantially the same” amino acid sequence isdefined as a sequence with at least 70%, preferably at least about 80%,more preferably at least about 85%, more preferably at least about 90%,even more preferably at least about 95%, and most preferably at least99% homology with another amino acid sequence, as determined by theFASTA search method in accordance with Pearson & Lipman, 1988, Proc.Natl. Inst. Acad. Sci. USA 85:2444-48.

By the term “specifically bind” or “specifically binds,” as used herein,is meant that a first molecule preferentially binds to a secondmolecule, but does not necessarily bind only to that second molecule.

As used herein, the terms “therapy” or “therapeutic regimen” refer tothose activities taken to alleviate or alter a disorder or diseasestate, e.g., a course of treatment intended to reduce or eliminate atleast one sign or symptom of a disease or disorder usingpharmacological, surgical, dietary and/or other techniques. Atherapeutic regimen may include a prescribed dosage of one or more drugsor surgery. Therapies will most often be beneficial and reduce oreliminate at least one sign or symptom of the disorder or disease state,but in some instances the effect of a therapy will have non-desirable orside-effects. The effect of therapy will also be impacted by thephysiological state of the subject, e.g., age, gender, genetics, weight,other disease conditions, etc.

The term “therapeutically effective amount” refers to the amount of thesubject compound that will elicit the biological or medical response ofa tissue, system, or subject that is being sought by the researcher,veterinarian, medical doctor or other clinician. The term“therapeutically effective amount” includes that amount of a compoundthat, when administered, is sufficient to prevent development of, oralleviate to some extent, one or more of the signs or symptoms of thedisorder or disease being treated. The therapeutically effective amountwill vary depending on the compound, the disease and its severity andthe age, weight, etc., of the subject to be treated.

To “treat” a disease as the term is used herein, means to reduce thefrequency or severity of at least one sign or symptom of a disease ordisorder experienced by a subject.

“Variant” as the term is used herein, is a nucleic acid sequence or apeptide sequence that differs in sequence from a reference nucleic acidsequence or peptide sequence respectively, but retains essentialproperties of the reference molecule. Changes in the sequence of anucleic acid variant may not alter the amino acid sequence of a peptideencoded by the reference nucleic acid, or may result in amino acidsubstitutions, additions, deletions, fusions and truncations. Changes inthe sequence of peptide variants are typically limited or conservative,so that the sequences of the reference peptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference peptide can differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A variant of anucleic acid or peptide can be a naturally occurring such as an allelicvariant, or can be a variant that is not known to occur naturally.Non-naturally occurring variants of nucleic acids and peptides may bemade by mutagenesis techniques or by direct synthesis.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

DESCRIPTION

The present invention relates to peptide analogs of PEDF and usesthereof. For example, in certain instances the invention providespeptides and peptide analogs based on smaller active fragments of P78, a44 residue peptide from PEDF (residues 78 to 121), wherein the peptides,including peptides and analogs, fragments, and derivatives thereof, ofthe invention exhibit desirable therapeutic properties. In oneembodiment, the invention provides compositions comprising peptides andanalogs, fragments, and derivatives thereof that exhibit one or more ofimproved efficacy, half-life, bioavailability, and the like compared tofull length PEDF. In one embodiment, the composition comprises a peptidecomprising butyric acid at the N-terminus. In one embodiment thecomposition comprises a peptide being Pegylated at the C-terminus. Forexample, in one embodiment, the peptide comprises Fmoc-NH-PEG₂-CH₂COOHat the C-terminus. In one embodiment, the invention provides isolatednucleic acids encoding the peptides disclosed herein.

The invention provides peptide analogs of PEDF useful for a wide varietyof applications. The invention also provides methods of using thepeptides as well as kits containing the peptides. The small peptides ofthe invention advantageously allow the relative ease of crossing tissuebarriers, of providing active doses, of ease of synthesis inreproducibly pure large-scale quantities, and fewer side effects,compared to full length PEDF.

In another embodiment, the invention provides methods of using thecompositions of the invention in any therapeutic or prophylactictreatment known in the art, or subsequently discovered, that have beenused with full-length PEDF protein. Such therapeutic applications aredisclosed in detail in, for instance, U.S. Pat. Nos. 6,451,763;6,288,024; 6,391,850; 6,670,333; 6,797,691; 6,919,309 and U.S. PatentPublication 20060008900, each of which is enclosed herein in itsentirety.

Compositions

The invention provides isolated peptides and nucleic acids encoding suchpeptides. Also provided are vectors and cells comprising an isolatednucleic acid of the invention. The peptides, including peptides andanalogs, fragments, and derivatives thereof are based on smaller activefragments of P78, a 44 residue peptide from PEDF (residues 78 to 121).In certain embodiments, the peptides of the invention are human.

Exemplary peptides of the present invention are illustrated in Table 1and are identified as SpxA1 and 81-1 to 81-20. SpxA1, also referred toherein as “Spx,” comprises amino acid residues 78 to 106 of PEDF. In oneembodiment, the amino acid sequence of SpxA is provided by SEQ ID NO:44. The amino acid sequences for peptides 81-1 through 81-20 areprovided by SEQ ID NOs: 1 through 20, respectively. In one embodiment,the composition comprises an isolated nucleic acid encoding a peptide ofthe invention. For example, in one embodiment, the composition comprisesan isolated nucleic acid encoding a peptide having an amino acidsequence of one of SEQ ID NOs 1 through 20 or SEQ ID NO: 44.

In one embodiment, the invention includes variants of the peptides ofthe invention. In one embodiment, variants differ fromnaturally-occurring peptides by conservative amino acid sequencedifferences or by modifications which do not affect sequence, or byboth. For example, conservative amino acid changes may be made, whichalthough they alter the primary sequence of the peptide, do not normallyalter its function. Conservative amino acid substitutions typicallyinclude substitutions within the following groups:

glycine, alanine;

valine, isoleucine, leucine;

aspartic acid, glutamic acid;

asparagine, glutamine;

serine, threonine;

lysine, arginine;

phenylalanine, tyrosine.

In one embodiment, the peptide of the invention comprises a peptidehaving at least 75% homology with a peptide listed in Table 1 (i.e.peptides 81-1 to 81-20). In one embodiment, the peptide of the inventioncomprises a peptide having at least 80% homology with a peptide listedin Table 1. In one embodiment, the peptide of the invention comprises apeptide having at least 85% homology with a peptide listed in Table 1.In one embodiment, the peptide of the invention comprises a peptidehaving at least 90% homology with a peptide listed in Table 1. In oneembodiment, the peptide of the invention comprises a peptide having atleast 95% homology with a peptide listed in Table 1. In one embodiment,the peptide of the invention comprises a peptide having at least 99%homology with a peptide listed in Table 1.

In a further embodiment, the peptide of the invention comprise D-, L-,and unnatural isomers of amino acids. In one embodiment, the compositioncomprises a peptide comprising one or more unnatural or non-naturalamino acids. Non-natural amino acids include, but are not limited to,the D-amino acids, 2,4-diaminobutyric acid, α-amino isobutyric acid,2-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid,g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid,3-amino propionic acid, ornithine, norleucine, norvaline,hydroxyproline, sarcosine, naphthalene, L-1-naphthalene, citrulline,homocitrulline, cysteic acid, t-butylglycine, t-butylalanine,phenylglycine, cyclohexylalanine, b-alanine, fluoro-amino acids,designer amino acids such as b-methyl amino acids, Ca-methyl aminoacids, Na-methyl amino acids, and amino acid analogs in general.

As known in the art the “similarity” between two peptides is determinedby comparing the amino acid sequence and its conserved amino acidsubstitutes of one polypeptide to a sequence of a second peptide.Variants are defined to include polypeptide sequences different from theoriginal sequence, preferably different from the original sequence inless than 40% of residues per segment of interest, more preferablydifferent from the original sequence in less than 25% of residues persegment of interest, more preferably different by less than 10% ofresidues per segment of interest, most preferably different from theoriginal protein sequence in just a few residues per segment of interestand at the same time sufficiently homologous to the original sequence topreserve the functionality of the original sequence and/or the abilityto bind to ubiquitin or to a ubiquitylated protein. The presentinvention includes amino acid sequences that are at least 60%, 65%, 70%,72%, 74%, 76%, 78%, 80%, 90%, or 95% similar or identical to theoriginal amino acid sequence. The degree of identity between twopolypeptides is determined using computer algorithms and methods thatare widely known for the persons skilled in the art. The identitybetween two amino acid sequences is preferably determined by using theBLASTP algorithm [BLAST Manual, Altschul, S., et al., NCBI NLM NIHBethesda, Md. 20894, Altschul, S., et al., J. Mol. Biol. 215: 403-410(1990)].

In certain embodiments, the peptides of the invention comprise anN-terminal and/or C-terminal modifications that in certain instancesimprove activity. For example, in one embodiment, the peptide of theinvention comprises a butyric acid at the N-terminus of the peptide. Inone embodiment, the peptide is PEGylated at the C-terminus of thepeptide. The present invention encompasses variants of the peptideanalogs, including those with terminal modifications, without terminalmodifications, or having different terminal modifications.

For example, in one embodiment, the composition of the inventioncomprises 81-12({BA*}NFGYDLYRVRSSMSPTTNSALSLGADERTESIIHR {PEG*}; SEQ IDNO:12), a peptide having a butyric acid at the N-terminus and PEGylatedat the C-terminus. However, the present invention also encompassesanalogs and derivatives of 81-12, including peptides having differentterminal modifications or no terminal modifications. That is, thepresent invention encompasses a composition comprising a peptidecomprising the amino acid sequence ofNFGYDLYRVRSSMSPTTNSALSLGADERTESIIHR (SEQ ID NO: 46), or an isolatednucleic acid encoding SEQ ID NO: 46. Similarly, the present inventionencompasses variants of Spx, and 81-1 to 81-20, including variants withalternative N-terminal and/or C-terminal modifications.

Variants of suitable peptides of the invention can also be expressed.Variants may be made by, for example, the deletion, addition, oralteration of amino acids that have either (i) minimal influence oncertain properties, secondary structure, and hydropathic nature of thepolypeptide or (ii) substantial effect on one or more properties of thepeptide mimetics of the invention.

Variants may also include, for example, a peptide conjugated to a linkeror other sequence for ease of synthesis, purification, identification,or therapeutic use (i.e., delivery) of the peptide.

The variants of the peptides according to the present invention may be(i) one in which one or more of the amino acid residues are substitutedwith a conserved or non-conserved amino acid residue (preferably aconserved amino acid residue) and such substituted amino acid residuemay or may not be one encoded by the genetic code, (ii) one in whichthere are one or more modified amino acid residues, e.g., residues thatare modified by the attachment of substituent groups, (iii) one in whichthe peptide is an alternative splice variant of the peptide of thepresent invention, (iv) fragments of the peptides and/or (v) one inwhich the peptide is fused with another peptide, such as a leader orsecretory sequence or a sequence which is employed for purification (forexample, His-tag) or for detection (for example, Sv5 epitope tag). Thefragments include peptides generated via proteolytic cleavage (includingmulti-site proteolysis) of an original sequence. Variants may bepost-translationally, or chemically modified. Such variants are deemedto be within the scope of those skilled in the art from the teachingherein.

The peptides of the invention can be post-translationally modified. Forexample, post-translational modifications that fall within the scope ofthe present invention include signal peptide cleavage, glycosylation,acetylation, isoprenylation, proteolysis, myristoylation, proteinfolding and proteolytic processing, etc. Some modifications orprocessing events require introduction of additional biologicalmachinery. For example, processing events, such as signal peptidecleavage and core glycosylation, are examined by adding caninemicrosomal membranes or Xenopus egg extracts (U.S. Pat. No. 6,103,489)to a standard translation reaction.

The peptides of the invention may include unnatural amino acids formedby post-translational modification or by introducing unnatural aminoacids during translation. A variety of approaches are available forintroducing unnatural amino acids during protein translation. By way ofexample, special tRNAs, such as tRNAs which have suppressor properties,suppressor tRNAs, have been used in the process of site-directednon-native amino acid replacement (SNAAR). In SNAAR, a unique codon isrequired on the mRNA and the suppressor tRNA, acting to target anon-native amino acid to a unique site during the protein synthesis(described in WO90/05785). However, the suppressor tRNA must not berecognizable by the aminoacyl tRNA synthetases present in the proteintranslation system. In certain cases, a non-native amino acid can beformed after the tRNA molecule is aminoacylated using chemical reactionswhich specifically modify the native amino acid and do not significantlyalter the functional activity of the aminoacylated tRNA. These reactionsare referred to as post-aminoacylation modifications. For example, theepsilon-amino group of the lysine linked to its cognate tRNA(tRNA_(LYS)), could be modified with an amine specific photoaffinitylabel.

The peptides of the invention may be conjugated with other molecules,such as proteins, to prepare fusion proteins. This may be accomplished,for example, by the synthesis of N-terminal or C-terminal fusionproteins provided that the resulting fusion protein retains thefunctionality of the peptide of the invention.

Cyclic derivatives of the peptides the invention are also part of thepresent invention. Cyclization may allow the peptide to assume a morefavorable conformation for association with other molecules. Cyclizationmay be achieved using techniques known in the art. For example,disulfide bonds may be formed between two appropriately spacedcomponents having free sulfhydryl groups, or an amide bond may be formedbetween an amino group of one component and a carboxyl group of anothercomponent. Cyclization may also be achieved using anazobenzene-containing amino acid as described by Ulysse, L., et al., J.Am. Chem. Soc. 1995, 117, 8466-8467. The components that form the bondsmay be side chains of amino acids, non-amino acid components or acombination of the two. In an embodiment of the invention, cyclicpeptides may comprise a beta-turn in the right position. Beta-turns maybe introduced into the peptides of the invention by adding the aminoacids Pro-Gly at the right position.

It may be desirable to produce a cyclic peptide which is more flexiblethan the cyclic peptides containing peptide bond linkages as describedabove. A more flexible peptide may be prepared by introducing cysteinesat the right and left position of the peptide and forming a disulphidebridge between the two cysteines. The two cysteines are arranged so asnot to deform the beta-sheet and turn. The peptide is more flexible as aresult of the length of the disulfide linkage and the smaller number ofhydrogen bonds in the beta-sheet portion. The relative flexibility of acyclic peptide can be determined by molecular dynamics simulations.

The peptides of the invention may be converted into pharmaceutical saltsby reacting with inorganic acids such as hydrochloric acid, sulfuricacid, hydrobromic acid, phosphoric acid, etc., or organic acids such asformic acid, acetic acid, propionic acid, glycolic acid, lactic acid,pyruvic acid, oxalic acid, succinic acid, malic acid, tartaric acid,citric acid, benzoic acid, salicylic acid, benezenesulfonic acid, andtoluenesulfonic acids.

Peptides of the invention may also have modifications. Modifications(which do not normally alter primary sequence) include in vivo, or invitro chemical derivatization of polypeptides, e.g., acetylation, orcarboxylation. Also included are modifications of glycosylation, e.g.,those made by modifying the glycosylation patterns of a polypeptideduring its synthesis and processing or in further processing steps;e.g., by exposing the polypeptide to enzymes which affect glycosylation,e.g., mammalian glycosylating or deglycosylating enzymes. Also embracedare sequences which have phosphorylated amino acid residues, e.g.,phosphotyrosine, phosphoserine, or phosphothreonine.

Also included are peptides which have been modified using ordinarymolecular biological techniques so as to improve their resistance toproteolytic degradation or to optimize solubility properties or torender them more suitable as a therapeutic agent. Such variants includethose containing residues other than naturally-occurring L-amino acids,e.g., D-amino acids or non-naturally-occurring synthetic amino acids.The peptides of the invention may further be conjugated to non-aminoacid moieties that are useful in their therapeutic application. Inparticular, moieties that improve the stability, biological half-life,water solubility, and/or immunologic characteristics of the peptide areuseful. A non-limiting example of such a moiety is polyethylene glycol(PEG).

Covalent attachment of biologically active compounds to water-solublepolymers is one method for alteration and control of biodistribution,pharmacokinetics, and often, toxicity for these compounds (Duncan etal., 1984, Adv. Polym. Sci. 57:53-101). Many water-soluble polymers havebeen used to achieve these effects, such as poly(sialic acid), dextran,poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA),poly(N-vinylpyrrolidone) (PVP), poly(vinyl alcohol) (PVA), poly(ethyleneglycol-co-propylene glycol), poly(N-acryloyl morpholine (PAcM), andpoly(ethylene glycol) (PEG) (Powell, 1980, Polyethylene glycol. In R. L.Davidson (Ed.) Handbook of Water Soluble Gums and Resins. McGraw-Hill,New York, chapter 18). PEG possess an ideal set of properties: very lowtoxicity (Pang, 1993, J. Am. Coll. Toxicol. 12: 429-456) excellentsolubility in aqueous solution (Powell, supra), low immunogenicity andantigenicity (Dreborg et al., 1990, Crit. Rev. Ther. Drug Carrier Syst.6: 315-365). PEG-conjugated or “PEGylated” protein therapeutics,containing single or multiple chains of polyethylene glycol on theprotein, have been described in the scientific literature (Clark et al.,1996, J. Biol. Chem. 271: 21969-21977; Hershfield, 1997, Biochemistryand immunology of poly(ethylene glycol)-modified adenosine deaminase(PEG-ADA). In J. M. Harris and S. Zalipsky (Eds) Poly(ethylene glycol):Chemistry and Biological Applications. American Chemical Society,Washington, D.C., p 145-154; Olson et al., 1997, Preparation andcharacterization of poly(ethylene glycol)ylated human growth hormoneantagonist. In J. M. Harris and S. Zalipsky (Eds) Poly(ethylene glycol):Chemistry and Biological Applications. American Chemical Society,Washington, D.C., p 170-181).

A peptide of the invention may be synthesized by conventionaltechniques. For example, the peptides of the invention may besynthesized by chemical synthesis using solid phase peptide synthesis.These methods employ either solid or solution phase synthesis methods(see for example, J. M. Stewart, and J. D. Young, Solid Phase PeptideSynthesis, 2^(nd) Ed., Pierce Chemical Co., Rockford Ill. (1984) and G.Barany and R. B. Merrifield, The Peptides: Analysis Synthesis, Biologyeditors E. Gross and J. Meienhofer Vol. 2 Academic Press, New York,1980, pp. 3-254 for solid phase synthesis techniques; and M Bodansky,Principles of Peptide Synthesis, Springer-Verlag, Berlin 1984, and E.Gross and J. Meienhofer, Eds., The Peptides: Analysis, Synthesis,Biology, suprs, Vol 1, for classical solution synthesis.)

The peptides may be chemically synthesized by Merrifield-type solidphase peptide synthesis. This method may be routinely performed to yieldpeptides up to about 60-70 residues in length, and may, in some cases,be utilized to make peptides up to about 100 amino acids long. Largerpeptides may also be generated synthetically via fragment condensationor native chemical ligation (Dawson et al., 2000, Ann. Rev. Biochem.69:923-960). An advantage to the utilization of a synthetic peptideroute is the ability to produce large amounts of peptides, even thosethat rarely occur naturally, with relatively high purities, i.e.,purities sufficient for research, diagnostic or therapeutic purposes.

Solid phase peptide synthesis is described by Stewart et al. in SolidPhase Peptide Synthesis, 2nd Edition, 1984, Pierce Chemical Company,Rockford, Ill.; and Bodanszky and Bodanszky in The Practice of PeptideSynthesis, 1984, Springer-Verlag, New York. At the outset, a suitablyprotected amino acid residue is attached through its carboxyl group to aderivatized, insoluble polymeric support, such as cross-linkedpolystyrene or polyamide resin. “Suitably protected” refers to thepresence of protecting groups on both the alpha-amino group of the aminoacid, and on any side chain functional groups. Side chain protectinggroups are generally stable to the solvents, reagents and reactionconditions used throughout the synthesis, and are removable underconditions which will not affect the final peptide product. Stepwisesynthesis of the oligopeptide is carried out by the removal of theN-protecting group from the initial amino acid, and coupling thereto ofthe carboxyl end of the next amino acid in the sequence of the desiredpeptide. This amino acid is also suitably protected. The carboxyl of theincoming amino acid can be activated to react with the N-terminus of thesupport-bound amino acid by formation into a reactive group, such asformation into a carbodiimide, a symmetric acid anhydride, or an “activeester” group, such as hydroxybenzotriazole or pentafluorophenyl esters.

Examples of solid phase peptide synthesis methods include the BOC methodwhich utilized tert-butyloxcarbonyl as the alpha-amino protecting group,and the FMOC method which utilizes 9-fluorenylmethyloxcarbonyl toprotect the alpha-amino of the amino acid residues, both which methodsare well-known by those of skill in the art.

Incorporation of N- and/or C-blocking groups may also be achieved usingprotocols conventional to solid phase peptide synthesis methods. Forincorporation of C-terminal blocking groups, for example, synthesis ofthe desired peptide is typically performed using, as solid phase, asupporting resin that has been chemically modified so that cleavage fromthe resin results in a peptide having the desired C-terminal blockinggroup. To provide peptides in which the C-terminus bears a primary aminoblocking group, for instance, synthesis is performed using ap-methylbenzhydrylamine (MBHA) resin, so that, when peptide synthesis iscompleted, treatment with hydrofluoric acid releases the desiredC-terminally amidated peptide. Similarly, incorporation of anN-methylamine blocking group at the C-terminus is achieved usingN-methylaminoethyl-derivatized DVB, resin, which upon HF treatmentreleases a peptide bearing an N-methylamidated C-terminus. Blockage ofthe C-terminus by esterification can also be achieved using conventionalprocedures. This entails use of resin/blocking group combination thatpermits release of side-chain peptide from the resin, to allow forsubsequent reaction with the desired alcohol, to form the esterfunction. FMOC protecting group, in combination with DVB resinderivatized with methoxyalkoxybenzyl alcohol or equivalent linker, canbe used for this purpose, with cleavage from the support being effectedby TFA in dicholoromethane. Esterification of the suitably activatedcarboxyl function, e.g. with DCC, can then proceed by addition of thedesired alcohol, followed by de-protection and isolation of theesterified peptide product.

In one embodiment, the peptides of the invention are manufactured bysolid phase peptide synthesis using Fmoc chemistry. In certainembodiments, after synthesis the Fmoc group is deprotected at theN-terminus, the side chain protection group is deprotected, and thepeptide is cleaved from the resin. In one embodiment, the resin is aCl-resin. In one embodiment, the condensation reaction reagent isDIC+HOBT. In one embodiment deprotection is done using Pip. In certainembodiments, the synthesized peptides are purified by RP-HPLC using asolvent of acetonitrile+deionized with TFA as the buffer. In oneembodiment, the peptides are purified by gradient elution.

The peptides of the invention may be prepared by standard chemical orbiological means of peptide synthesis. Biological methods include,without limitation, expression of a nucleic acid encoding a peptide in ahost cell or in an in vitro translation system.

Included in the invention are nucleic acid sequences that encode thepeptide of the invention. In one embodiment, the invention includesnucleic acid sequences corresponding to the amino acid sequences of anyone of the peptides listed in Table 1. Accordingly, subclones of anucleic acid sequence encoding a peptide of the invention can beproduced using conventional molecular genetic manipulation forsubcloning gene fragments, such as described by Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory, ColdSprings Harbor, New York (2012), and Ausubel et al. (ed.), CurrentProtocols in Molecular Biology, John Wiley & Sons (New York, N.Y.) (1999and preceding editions), each of which is hereby incorporated byreference in its entirety. The subclones then are expressed in vitro orin vivo in bacterial cells to yield a smaller protein or polypeptidethat can be tested for a particular activity.

Biological preparation of a peptide of the invention involves expressionof a nucleic acid encoding a desired peptide. An expression cassettecomprising such a coding sequence may be used to produce a desiredpeptide for use in the method of the invention.

In the context of an expression vector, the vector can be readilyintroduced into a host cell, e.g., mammalian, bacterial, yeast or insectcell by any method in the art. Coding sequences for a desired peptide ofthe invention may be codon optimized based on the codon usage of theintended host cell in order to improve expression efficiency asdemonstrated herein. Codon usage patterns can be found in the literature(Nakamura et al., 2000, Nuc Acids Res. 28:292). Representative examplesof appropriate hosts include bacterial cells, such as streptococci,staphylococci, E. coli, Streptomyces and Bacillus subtilis cells; fungalcells, such as yeast cells and Aspergillus cells; insect cells such asDrosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS,HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells; and plant cells.

Numerous vectors are known in the art including, but not limited to,linear polynucleotides, polynucleotides associated with ionic oramphiphilic compounds, plasmids, and viruses. Thus, the term “vector”includes an autonomously replicating plasmid or a virus. The term shouldalso be construed to include non-plasmid and non-viral compounds whichfacilitate transfer of nucleic acid into cells, such as, for example,polylysine compounds, liposomes, and the like. Examples of viral vectorsinclude, but are not limited to, adenoviral vectors, adeno-associatedvirus vectors, retroviral vectors, and the like.

The expression vector can be transferred into a host cell by physical,biological or chemical means, discussed in detail elsewhere herein.

Examples of biological methods to prepare the peptides of the presentinvention may utilize methods provided in published US Patentapplication number US 2009/0069241, which is incorporated herein in itsentirety.

To ensure that the peptide obtained from either chemical or biologicalsynthetic techniques is the desired peptide, analysis of the peptidecomposition can be conducted. Such amino acid composition analysis maybe conducted using high resolution mass spectrometry to determine themolecular weight of the peptide. Alternatively, or additionally, theamino acid content of the peptide can be confirmed by hydrolyzing thepeptide in aqueous acid, and separating, identifying and quantifying thecomponents of the mixture using HPLC, or an amino acid analyzer. Proteinsequenators, which sequentially degrade the peptide and identify theamino acids in order, may also be used to determine definitely thesequence of the peptide.

In one embodiment, the present invention provides a compositioncomprising an isolated nucleic acid encoding a PEDF derived peptide, ora fragment thereof. In one embodiment, the isolated nucleic acid encodesa peptide comprising an amino acid sequence selected from SEQ ID NO: 44and SEQ ID NOs: 1-20. Further, the invention encompasses an isolatednucleic acid encoding a peptide having substantial homology to a peptidedisclosed herein. In certain embodiments, the isolated nucleic acidsequence encodes a peptide having at least 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99% sequence homology with an amino acid sequence selectedfrom SEQ ID NO: 44 and SEQ ID NOs: 1-20.

The nucleic acid sequence encoding the SpxA1 peptide is provided SEQ IDNO: 45. Nucleic acid sequences encoding peptides 81-1 to 81-20, orportions thereof, are provided by SEQ ID NOs 21-41. The nucleic acidsequence encoding peptide 81-1 is provided SEQ ID NO: 21. The nucleicacid sequence encoding peptide 81-2 is provided SEQ ID NO: 22. Thenucleic acid sequence encoding peptide 81-3 is provided SEQ ID NO: 23.The nucleic acid sequence encoding peptide 81-4 is provided SEQ ID NO:24. The nucleic acid sequence encoding peptide 81-5 is provided SEQ IDNO: 25. The nucleic acid sequence encoding peptide 81-6 is provided SEQID NO: 26. The nucleic acid sequence encoding peptide 81-7 is providedSEQ ID NO: 27. The nucleic acid sequence encoding peptide 81-8 isprovided SEQ ID NO: 28. The nucleic acid sequence encoding peptide 81-9is provided SEQ ID NO: 29. The nucleic acid sequence encoding peptide81-10 is provided SEQ ID NO: 30. The nucleic acid sequence encodingpeptide 81-11 is provided SEQ ID NO: 31. The nucleic acid sequenceencoding peptide 81-12 is provided SEQ ID NO: 32. The nucleic acidsequence encoding peptide 81-13 is provided SEQ ID NO: 13. The nucleicacid sequence encoding peptide 81-14 is provided SEQ ID NO: 34. Thenucleic acid sequence encoding peptide 81-15 is provided SEQ ID NO: 35.The nucleic acid sequence encoding peptide 81-16 is provided SEQ ID NO:36. The nucleic acid sequence encoding peptide 81-17 is provided SEQ IDNO: 37. The nucleic acid sequences encoding peptide 81-18 is providedSEQ ID NO: 38 and SEQ ID NO: 39. The nucleic acid sequence encodingpeptide 81-19 is provided SEQ ID NO: 40. The nucleic acid sequenceencoding peptide 81-20 is provided SEQ ID NO: 41.

For example, in one embodiment, the isolated nucleic acid comprises anucleotide sequence selected from SEQ ID NOs: 21-41. Further, theinvention encompasses an isolated nucleic acid having substantialhomology to a nucleic acid disclosed herein. In certain embodiments, theisolated nucleic acid has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% sequence homology with a nucleotide sequence selected fromSEQ ID NO: 45 and SEQ ID NOs: 21-41.

The isolated nucleic acid may comprise any type of nucleic acid,including, but not limited to DNA and RNA. For example, in oneembodiment, the composition comprises an isolated DNA molecule,including for example, an isolated cDNA molecule, encoding a peptide ofthe invention, or functional fragment thereof. In one embodiment, thecomposition comprises an isolated RNA molecule encoding a peptide of theinvention, or a functional fragment thereof. The isolated nucleic acidsmay be synthesized using any method known in the art.

The nucleic acid molecules of the present invention can be modified toimprove stability in serum or in growth medium for cell cultures.Modifications can be added to enhance stability, functionality, and/orspecificity and to minimize immunostimulatory properties of the nucleicacid molecule of the invention. For example, in order to enhance thestability, the 3′-residues may be stabilized against degradation, e.g.,they may be selected such that they consist of purine nucleotides,particularly adenosine or guanosine nucleotides. Alternatively,substitution of pyrimidine nucleotides by modified analogues, e.g.,substitution of uridine by 2′-deoxythymidine is tolerated and does notaffect function of the molecule.

In one embodiment of the present invention the nucleic acid molecule maycontain at least one modified nucleotide analogue. For example, the endsmay be stabilized by incorporating modified nucleotide analogues.

Non-limiting examples of nucleotide analogues include sugar- and/orbackbone-modified ribonucleotides (i.e., include modifications to thephosphate-sugar backbone). For example, the phosphodiester linkages ofnatural RNA may be modified to include at least one of a nitrogen orsulfur heteroatom. In preferred backbone-modified ribonucleotides thephosphoester group connecting to adjacent ribonucleotides is replaced bya modified group, e.g., of phosphothioate group. In preferredsugar-modified ribonucleotides, the 2′ OH-group is replaced by a groupselected from H, OR, R, halo, SH, SR, NH₂, NHR, NR₂ or ON, wherein R isC₁-C₆ alkyl, alkenyl or alkynyl and halo is F, Cl, Br or I.

Other examples of modifications are nucleobase-modified ribonucleotides,i.e., ribonucleotides, containing at least one non-naturally occurringnucleobase instead of a naturally occurring nucleobase. Bases may bemodified to block the activity of adenosine deaminase. Exemplarymodified nucleobases include, but are not limited to, uridine and/orcytidine modified at the 5-position, e.g., 5-(2-amino)propyl uridine,5-bromo uridine; adenosine and/or guanosines modified at the 8 position,e.g., 8-bromo guanosine; deaza nucleotides, e.g., 7-deaza-adenosine; O-and N-alkylated nucleotides, e.g., N6-methyl adenosine are suitable. Itshould be noted that the above modifications may be combined.

In some instances, the nucleic acid molecule comprises at least one ofthe following chemical modifications: 2′-H, 2′-O-methyl, or 2′-OHmodification of one or more nucleotides. In certain embodiments, anucleic acid molecule of the invention can have enhanced resistance tonucleases. For increased nuclease resistance, a nucleic acid molecule,can include, for example, 2′-modified ribose units and/orphosphorothioate linkages. For example, the 2′ hydroxyl group (OH) canbe modified or replaced with a number of different “oxy” or “deoxy”substituents. For increased nuclease resistance the nucleic acidmolecules of the invention can include 2′-O-methyl, 2′-fluorine,2′-O-methoxyethyl, 2′-O-aminopropyl, 2′-amino, and/or phosphorothioatelinkages. Inclusion of locked nucleic acids (LNA), ethylene nucleicacids (ENA), e.g., 2′-4′-ethylene-bridged nucleic acids, and certainnucleobase modifications such as 2-amino-A, 2-thio (e.g., 2-thio-U),G-clamp modifications, can also increase binding affinity to a target.

In one embodiment, the nucleic acid molecule includes a 2′-modifiednucleotide, e.g., a 2′-deoxy, 2′-deoxy-2′-fluoro, 2′-O-methyl,2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl (2′-O-AP),2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl(2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or2′-O-N-methylacetamido (2′-O-NMA). In one embodiment, the nucleic acidmolecule includes at least one 2′-O-methyl-modified nucleotide, and insome embodiments, all of the nucleotides of the nucleic acid moleculeinclude a 2′-O-methyl modification.

Nucleic acid agents discussed herein include otherwise unmodified RNAand DNA as well as RNA and DNA that have been modified, e.g., to improveefficacy, and polymers of nucleoside surrogates. Unmodified RNA refersto a molecule in which the components of the nucleic acid, namelysugars, bases, and phosphate moieties, are the same or essentially thesame as that which occur in nature, preferably as occur naturally in thehuman body. The art has referred to rare or unusual, but naturallyoccurring, RNAs as modified RNAs, see, e.g., Limbach et al. (NucleicAcids Res., 1994, 22:2183-2196). Such rare or unusual RNAs, often termedmodified RNAs, are typically the result of a post-transcriptionalmodification and are within the term unmodified RNA as used herein.Modified RNA, as used herein, refers to a molecule in which one or moreof the components of the nucleic acid, namely sugars, bases, andphosphate moieties, are different from that which occur in nature,preferably different from that which occurs in the human body. Whilethey are referred to as “modified RNAs” they will of course, because ofthe modification, include molecules that are not, strictly speaking,RNAs. Nucleoside surrogates are molecules in which the ribophosphatebackbone is replaced with a non-ribophosphate construct that allows thebases to be presented in the correct spatial relationship such thathybridization is substantially similar to what is seen with aribophosphate backbone, e.g., non-charged mimics of the ribophosphatebackbone.

Modifications of the nucleic acid of the invention may be present at oneor more of, a phosphate group, a sugar group, backbone, N-terminus,C-terminus, or nucleobase.

Vectors

The present invention also includes a vector in which the isolatednucleic acid of the present invention is inserted. The art is repletewith suitable vectors that are useful in the present invention.

In brief summary, the expression of natural or synthetic nucleic acidsencoding a peptide is typically achieved by operably linking a nucleicacid encoding the peptide or portions thereof to a promoter, andincorporating the construct into an expression vector. The vectors to beused are suitable for replication and, optionally, integration ineukaryotic cells. Typical vectors contain transcription and translationterminators, initiation sequences, and promoters useful for regulationof the expression of the desired nucleic acid sequence.

The vectors of the present invention may also be used for nucleic acidimmunization and gene therapy, using standard gene delivery protocols.Methods for gene delivery are known in the art. See, e.g., U.S. Pat.Nos. 5,399,346, 5,580,859, 5,589,466, incorporated by reference hereinin their entireties. In another embodiment, the invention provides agene therapy vector.

The isolated nucleic acid of the invention can be cloned into a numberof types of vectors. For example, the nucleic acid can be cloned into avector including, but not limited to a plasmid, a phagemid, a phagederivative, an animal virus, and a cosmid. Vectors of particularinterest include expression vectors, replication vectors, probegeneration vectors, and sequencing vectors.

Further, the vector may be provided to a cell in the form of a viralvector. Viral vector technology is well known in the art and isdescribed, for example, in Sambrook et al. (2001, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, New York), and inother virology and molecular biology manuals. Viruses, which are usefulas vectors include, but are not limited to, retroviruses, adenoviruses,adeno- associated viruses, herpes viruses, and lentiviruses. In general,a suitable vector contains an origin of replication functional in atleast one organism, a promoter sequence, convenient restrictionendonuclease sites, and one or more selectable markers, (e.g., WO01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).

A number of viral based systems have been developed for gene transferinto mammalian cells. For example, retroviruses provide a convenientplatform for gene delivery systems. A selected gene can be inserted intoa vector and packaged in retroviral particles using techniques known inthe art. The recombinant virus can then be isolated and delivered tocells of the subject either in vivo or ex vivo. A number of retroviralsystems are known in the art. In some embodiments, adenovirus vectorsare used. A number of adenovirus vectors are known in the art. In oneembodiment, lentivirus vectors are used.

For example, vectors derived from retroviruses such as the lentivirusare suitable tools to achieve long-term gene transfer since they allowlong-term, stable integration of a transgene and its propagation indaughter cells. Lentiviral vectors have the added advantage over vectorsderived from onco-retroviruses such as murine leukemia viruses in thatthey can transduce non-proliferating cells, such as hepatocytes. Theyalso have the added advantage of low immunogenicity. In one embodiment,the composition includes a vector derived from an adeno-associated virus(AAV). Adeno-associated viral (AAV) vectors have become powerful genedelivery tools for the treatment of various disorders. AAV vectorspossess a number of features that render them ideally suited for genetherapy, including a lack of pathogenicity, minimal immunogenicity, andthe ability to transduce postmitotic cells in a stable and efficientmanner. Expression of a particular gene contained within an AAV vectorcan be specifically targeted to one or more types of cells by choosingthe appropriate combination of AAV serotype, promoter, and deliverymethod

In certain embodiments, the vector also includes conventional controlelements which are operably linked to the transgene in a manner whichpermits its transcription, translation and/or expression in a celltransfected with the plasmid vector or infected with the virus producedby the invention. As used herein, “operably linked” sequences includeboth expression control sequences that are contiguous with the gene ofinterest and expression control sequences that act in trans or at adistance to control the gene of interest. Expression control sequencesinclude appropriate transcription initiation, termination, promoter andenhancer sequences; efficient RNA processing signals such as splicingand polyadenylation (polyA) signals; sequences that stabilizecytoplasmic mRNA; sequences that enhance translation efficiency (i.e.,Kozak consensus sequence); sequences that enhance protein stability; andwhen desired, sequences that enhance secretion of the encoded product. Agreat number of expression control sequences, including promoters whichare native, constitutive, inducible and/or tissue-specific, are known inthe art and may be utilized.

Additional promoter elements, e.g., enhancers, regulate the frequency oftranscriptional initiation. Typically, these are located in the region30-110 bp upstream of the start site, although a number of promotershave recently been shown to contain functional elements downstream ofthe start site as well. The spacing between promoter elements frequentlyis flexible, so that promoter function is preserved when elements areinverted or moved relative to one another. In the thymidine kinase (tk)promoter, the spacing between promoter elements can be increased to 50bp apart before activity begins to decline. Depending on the promoter,it appears that individual elements can function either cooperatively orindependently to activate transcription.

One example of a suitable promoter is the immediate earlycytomegalovirus (CMV) promoter sequence. This promoter sequence is astrong constitutive promoter sequence capable of driving high levels ofexpression of any polynucleotide sequence operatively linked thereto.Another example of a suitable promoter is Elongation Growth Factor -1α(EF-1α). However, other constitutive promoter sequences may also beused, including, but not limited to the simian virus 40 (SV40) earlypromoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus(HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avianleukemia virus promoter, an Epstein-Barr virus immediate early promoter,a Rous sarcoma virus promoter, as well as human gene promoters such as,but not limited to, the actin promoter, the myosin promoter, thehemoglobin promoter, and the creatine kinase promoter. Further, theinvention should not be limited to the use of constitutive promoters.Inducible promoters are also contemplated as part of the invention. Theuse of an inducible promoter provides a molecular switch capable ofturning on expression of the polynucleotide sequence which it isoperatively linked when such expression is desired, or turning off theexpression when expression is not desired. Examples of induciblepromoters include, but are not limited to a metallothionine promoter, aglucocorticoid promoter, a progesterone promoter, and a tetracyclinepromoter.

Enhancer sequences found on a vector also regulates expression of thegene contained therein. Typically, enhancers are bound with proteinfactors to enhance the transcription of a gene. Enhancers may be locatedupstream or downstream of the gene it regulates. Enhancers may also betissue-specific to enhance transcription in a specific cell or tissuetype. In one embodiment, the vector of the present invention comprisesone or more enhancers to boost transcription of the gene present withinthe vector.

In order to assess the expression of the peptide, the expression vectorto be introduced into a cell can also contain either a selectable markergene or a reporter gene or both to facilitate identification andselection of expressing cells from the population of cells sought to betransfected or infected through viral vectors. In other aspects, theselectable marker may be carried on a separate piece of DNA and used ina co-transfection procedure. Both selectable markers and reporter genesmay be flanked with appropriate regulatory sequences to enableexpression in the host cells. Useful selectable markers include, forexample, antibiotic-resistance genes, such as neo and the like.

Reporter genes are used for identifying potentially transfected cellsand for evaluating the functionality of regulatory sequences. Ingeneral, a reporter gene is a gene that is not present in or expressedby the recipient organism or tissue and that encodes a polypeptide whoseexpression is manifested by some easily detectable property, e.g.,enzymatic activity. Expression of the reporter gene is assayed at asuitable time after the DNA has been introduced into the recipientcells. Suitable reporter genes may include genes encoding luciferase,beta-galactosidase, chloramphenicol acetyl transferase, secretedalkaline phosphatase, or the green fluorescent protein gene (e.g.,Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expressionsystems are well known and may be prepared using known techniques orobtained commercially. In general, the construct with the minimal 5′flanking region showing the highest level of expression of reporter geneis identified as the promoter. Such promoter regions may be linked to areporter gene and used to evaluate agents for the ability to modulatepromoter-driven transcription.

Methods of introducing and expressing genes into a cell are known in theart. In the context of an expression vector, the vector can be readilyintroduced into a host cell, e.g., mammalian, bacterial, yeast, orinsect cell by any method in the art. For example, the expression vectorcan be transferred into a host cell by physical, chemical, or biologicalmeans.

Physical methods for introducing a polynucleotide into a host cellinclude calcium phosphate precipitation, lipofection, particlebombardment, microinjection, electroporation, and the like. Methods forproducing cells comprising vectors and/or exogenous nucleic acids arewell-known in the art. See, for example, Sambrook et al. (2012,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,New York). A preferred method for the introduction of a polynucleotideinto a host cell is calcium phosphate transfection.

Biological methods for introducing a polynucleotide of interest into ahost cell include the use of DNA and RNA vectors. Viral vectors, andespecially retroviral vectors, have become the most widely used methodfor inserting genes into mammalian, e.g., human cells. Other viralvectors can be derived from lentivirus, poxviruses, herpes simplex virusI, adenoviruses and adeno-associated viruses, and the like. See, forexample, U.S. Pat. Nos. 5,350,674 and 5,585,362.

Chemical means for introducing a polynucleotide into a host cell includecolloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Anexemplary colloidal system for use as a delivery vehicle in vitro and invivo is a liposome (e.g., an artificial membrane vesicle).

In the case where a non-viral delivery system is utilized, an exemplarydelivery vehicle is a liposome. The use of lipid formulations iscontemplated for the introduction of the nucleic acids into a host cell(in vitro, ex vivo or in vivo). In another aspect, the nucleic acid maybe associated with a lipid. The nucleic acid associated with a lipid maybe encapsulated in the aqueous interior of a liposome, interspersedwithin the lipid bilayer of a liposome, attached to a liposome via alinking molecule that is associated with both the liposome and theoligonucleotide, entrapped in a liposome, complexed with a liposome,dispersed in a solution containing a lipid, mixed with a lipid, combinedwith a lipid, contained as a suspension in a lipid, contained orcomplexed with a micelle, or otherwise associated with a lipid. Lipid,lipid/DNA or lipid/expression vector associated compositions are notlimited to any particular structure in solution. For example, they maybe present in a bilayer structure, as micelles, or with a “collapsed”structure. They may also simply be interspersed in a solution, possiblyforming aggregates that are not uniform in size or shape. Lipids arefatty substances which may be naturally occurring or synthetic lipids.For example, lipids include the fatty droplets that naturally occur inthe cytoplasm as well as the class of compounds which contain long-chainaliphatic hydrocarbons and their derivatives, such as fatty acids,alcohols, amines, amino alcohols, and aldehydes.

Lipids suitable for use can be obtained from commercial sources. Forexample, dimyristyl phosphatidylcholine (“DMPC”) can be obtained fromSigma, St. Louis, Mo.; dicetyl phosphate (“DCP”) can be obtained from K& K Laboratories (Plainview, N.Y.); cholesterol (“Choi”) can be obtainedfrom Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) andother lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham,Ala.). Stock solutions of lipids in chloroform or chloroform/methanolcan be stored at about −20° C. Chloroform is used as the only solventsince it is more readily evaporated than methanol. “Liposome” is ageneric term encompassing a variety of single and multilamellar lipidvehicles formed by the generation of enclosed lipid bilayers oraggregates. Liposomes can be characterized as having vesicularstructures with a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh et al.,1991 Glycobiology 5: 505-10). However, compositions that have differentstructures in solution than the normal vesicular structure are alsoencompassed. For example, the lipids may assume a micellar structure ormerely exist as nonuniform aggregates of lipid molecules. Alsocontemplated are lipofectamine-nucleic acid complexes.

Regardless of the method used to introduce exogenous nucleic acids intoa host cell, in order to confirm the presence of the recombinant DNAsequence in the host cell, a variety of assays may be performed. Suchassays include, for example, “molecular biological” assays well known tothose of skill in the art, such as Southern and Northern blotting,RT-PCR and PCR; “biochemical” assays, such as detecting the presence orabsence of a particular peptide, e.g., by immunological means (ELISAsand Western blots) or by assays described herein to identify agentsfalling within the scope of the invention.

The present invention includes a composition comprising a cell whichcomprises a peptide of the invention, a nucleic acid encoding a peptideof the invention, or a combination thereof In one embodiment, the cellis genetically modified to comprise a peptide and/or nucleic acid of theinvention. In certain embodiments, genetically modified cell isautologous to a subject being treated with the composition of theinvention. Alternatively, the cells can be allogeneic, syngeneic, orxenogeneic with respect to the subject. In certain embodiment, the cellis able to secrete or release the expressed peptide of the inventioninto extracellular space in order to deliver the peptide to one or moreother cells.

The genetically modified cell may be modified in vivo or ex vivo, usingtechniques standard in the art. Genetic modification of the cell may becarried out using an expression vector or using a naked isolated nucleicacid construct.

In one embodiment, the cell is obtained and modified ex vivo, using anisolated nucleic acid encoding a peptide. In one embodiment, the cell isobtained from a subject, genetically modified to express the peptideand/or nucleic acid, and is re-administered to the subject. In certainembodiments, the cell is expanded ex vivo or in vitro to produce apopulation of cells, wherein at least a portion of the population isadministered to a subject in need.

In one embodiment, the cell is genetically modified to stably expressthe peptide. In another embodiment, the cell is genetically modified totransiently express the peptide.

The present invention provides a scaffold or substrate compositioncomprising a peptide of the invention, an isolated nucleic acid of theinvention, a cell comprising the peptide of the invention, or acombination thereof. For example, in one embodiment, a peptide of theinvention, an isolated nucleic acid of the invention, a cell producingthe peptide of the invention, or a combination thereof is incorporatedwithin a scaffold. In another embodiment, a peptide of the invention, anisolated nucleic acid of the invention, a cell producing the peptide ofthe invention, or a combination thereof is applied to the surface of ascaffold. The scaffold of the invention may be of any type known in theart. Non-limiting examples of such a scaffold includes a, hydrogel,electrospun scaffold, foam, mesh, sheet, patch, and sponge.

Methods

The peptides disclosed herein are useful in any method known in the art,or subsequently discovered, where PEDF protein is useful. For example,the peptides of the invention and nucleic acids encoding the peptidesmay be used in biological assays, screening assays, therapeutictreatments, prophylactic treatments, culture media supplements, and thelike.

Assays

In one embodiment, the peptides of the invention are used in abiological assay. For example, the one or more of the peptides of theinvention may be used to screen for tumor growth, apoptosis,cytoskeletal function, function of mitochondrial and other organelles,oxidative stress, inflammation, angiogenesis, neurogenesis, cell growth,immune function, cell differentiation, adipogenesis, and bonedeposition. In certain embodiments, the peptides are used to evaluatethe level of expression or activity of one or more members of the PEDFsignaling pathway in a cell or sample.

In certain embodiments, the present invention comprises an assaycomprising contacting a cell with one or more peptides disclosed herein.For example, the method may comprise administering the one or morepeptides or administering one or more isolated nucleic acids encodingthe one or more peptides. In certain embodiments, the method comprisescontacting the cell with a culture medium, buffer, or the likecomprising one or more peptides. In one embodiment, the method comprisesgenetically modifying a cell to express, and in certain instancessecrete, one or more of the peptides disclosed herein by contacting thecell with one or more isolated nucleic acids encoding the one or morepeptides. In one embodiment, the genetically modified cell is the cellbeing assayed. In one embodiment, the genetically modified cell isanother cell (i.e., a second cell). For example, in one embodiment, thegenetically modified cell may be a cell co-cultured with the cell beingassayed.

The present invention also includes assays for inflammation, cell deathor vascular leakage, comprising incubating cells with a peptide of thepresent invention and determining the effect of said peptide on saidcells. Such assays are useful to determine which peptide is the optimalchoice for use in treating a given disease state. Examples of suchassays are presented below in the Examples section.

In one aspect, the present invention is directed to a screening assay toidentify compounds that promote or inhibit PEDF signaling activity. Forexample, the screening assay can be used to identify compounds thatincrease or decrease the expression or activity of one or more membersof the PEDF signaling pathway, thereby altering PEDF signaling. Forexample, the screening assay may be used to identify compounds thatinfluence tumor growth, apoptosis, cytoskeletal function, function ofmitochondrial and other organelles, oxidative stress, inflammation,angiogenesis, neurogenesis, cell growth, immune function, celldifferentiation, adipogenesis, or bone deposition.

In one embodiment, the method comprises contacting a cell or biologicalsample with a peptide of the invention in the presence or absence of atest agent. The test agents can be obtained using any of the numerousapproaches in combinatorial-library methods known in the art, including:biological libraries; spatially addressable parallel solid phase orsolution phase libraries; synthetic library methods requiringdeconvolution; the “one-bead one-compound” library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary approach is limited to peptide libraries, while the other fourapproaches are applicable to peptide, non-peptide oligomer or smallmolecule libraries of compounds (Lam et al., 1997, Anticancer Drug Des.12:45).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example, in: DeWitt et al., 1993, Proc. Natl.Acad. USA 90:6909; Erb et al., 1994, Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al., 1994, J. Med. Chem. 37:2678; Cho et al.,1993, Science 261:1303; Carrell et al., 1994, Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al., 1994, Angew. Chem. Int. Ed. Engl. 33:2061;and Gallop et al., 1994, J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten,1992, Biotechniques 13:412-421), or on beads (Lam, 1991, Nature354:82-84), chips (Fodor, 1993, Nature 364:555-556), bacteria (LadnerU.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409), plasmids(Cull et al., 1992, Proc. Natl. Acad. Sci. USA 89:1865-1869) or on phage(Scott and Smith, 1990, Science 249:386-390; Devlin, 1990, Science249:404-406; Cwirla et al., 1990, Proc. Natl. Acad. Sci. USA87:6378-6382; Felici, 1991, J. Mol. Biol. 222:301-310; and Ladnersupra).

In situations where “high-throughput” modalities are preferred, it istypical that new chemical entities with useful properties are generatedby identifying a chemical compound (called a “lead compound”) with somedesirable property or activity, creating variants of the lead compound,and evaluating the property and activity of those variant compounds. Thecurrent trend is to shorten the time scale for all aspects of drugdiscovery.

In one embodiment, high throughput screening methods involve providing alibrary containing a large number of compounds (candidate compounds)potentially having the desired activity. Such “combinatorial chemicallibraries” are then screened in one or more assays, as described herein,to identify those library members (particular chemical species orsubclasses) that display a desired characteristic activity. Thecompounds thus identified can serve as conventional “lead compounds” orcan themselves be used as potential or actual therapeutics.

Antibodies

In one embodiment, the peptides of the present invention may be used forthe generation of an antibody. For example, one or more peptides of theinvention may be used to generate an antibody that specifically binds tothe peptide and therefore also to a region of PEDF.

Methods of making and using antibodies are well known in the art. Forexample, polyclonal antibodies are generated by immunizing rabbitsaccording to standard immunological techniques well-known in the art(see, e.g., Harlow et al., 1988, In: Antibodies, A Laboratory Manual,Cold Spring Harbor, N.Y.). Such techniques include immunizing an animalwith a chimeric protein comprising a portion of another protein such asa maltose binding protein or glutathione (GSH) tag polypeptide portion,and/or a moiety such that the antigenic protein of interest is renderedimmunogenic (e.g., an antigen of interest conjugated with keyhole limpethemocyanin, KLH) and a portion comprising the respective antigenicprotein amino acid residues. In the present invention, the peptides ofthe invention may serve as the antigen. The chimeric proteins areproduced by cloning the appropriate nucleic acids encoding the markerprotein into a plasmid vector suitable for this purpose, such as but notlimited to, pMAL-2 or pCMX.

The present invention should be construed to encompass antibodies whichbind to the specific antigens of interest (i.e. peptide analogs ofPEDF), and are able to bind the antigen present on Western blots, insolution in enzyme linked immunoassays, in fluorescence activated cellssorting (FACS) assays, in magenetic-actived cell sorting (MACS) assays,immunocytochemistry, immunoprecipitation, and in immunofluorescencemicroscopy of a cell transiently transfected with a nucleic acidencoding at least a portion of the antigenic protein, for example.

The antibodies of the invention may be used to neutralize the activityor to inhibit the activity of PEDF. For example, an antibody generatedwith the use of the peptides of the invention may be used as atherapeutic composition in order to inhibit the activity of PEDF, inconditions where excessive PEDF expression or activity is deleterious orassociated with a particular disease or disorder.

The antibodies can be produced by immunizing an animal such as, but notlimited to, a rabbit, a mouse or a camel, with an antigenic peptide ofthe invention, or a portion thereof, by immunizing an animal using aprotein comprising at least a portion of the peptide, or a fusionprotein including a tag polypeptide portion comprising, for example, amaltose binding protein tag polypeptide portion, covalently linked witha portion comprising the appropriate amino acid residues. One skilled inthe art would appreciate, based upon the disclosure provided herein,that smaller fragments of these peptide can also be used to produceantibodies that specifically bind the antigen of interest.

Once armed with the sequence of a specific antigen of interest and thedetailed analysis localizing the various conserved and non-conserveddomains of the protein, the skilled artisan would understand, based uponthe disclosure provided herein, how to obtain antibodies specific forthe various portions of the antigen using methods well-known in the artor to be developed.

Further, the skilled artisan, based upon the disclosure provided herein,would appreciate that using a non-conserved immunogenic portion canproduce antibodies specific for the non-conserved region therebyproducing antibodies that do not cross-react with other proteins whichcan share one or more conserved portions. Thus, one skilled in the artwould appreciate, based upon the disclosure provided herein, that thenon-conserved regions of an antigen of interest can be used to produceantibodies that are specific only for that antigenic peptide and do notcross-react non-specifically with other proteins or peptides, includingother types of PEDF peptide fragments.

The invention encompasses monoclonal, synthetic antibodies, and thelike. One skilled in the art would understand, based upon the disclosureprovided herein, that the crucial feature of the antibody of theinvention is that the antibody bind specifically with an antigen ofinterest. That is, the antibody of the invention recognizes an antigenof interest or a fragment thereof (e.g., an immunogenic portion orantigenic determinant thereof), on Western blots, in immunostaining ofcells, and immunoprecipitates the antigen using standard methodswell-known in the art.

One skilled in the art would appreciate, based upon the disclosureprovided herein, that the antibodies can be used to immunoprecipitateand/or immuno-affinity purify their cognate antigen as described indetail elsewhere herein, and additionally, by using methods well-knownin the art.

The skilled artisan would appreciate, based upon the disclosure providedherein, that that present invention includes use of a single antibodyrecognizing a single antigenic epitope but that the invention is notlimited to use of a single antibody. Instead, the invention encompassesuse of at least one antibody where the antibodies can be directed to thesame or different antigenic protein epitopes.

The generation of polyclonal antibodies is accomplished by inoculatingthe desired animal with the antigen and isolating antibodies whichspecifically bind the antigen therefrom using standard antibodyproduction methods such as those described in, for example, Harlow etal. (1988, In: Antibodies, A Laboratory Manual, Cold Spring Harbor,N.Y.).

Monoclonal antibodies directed against full length or peptide fragmentsof a protein or peptide may be prepared using any well-known monoclonalantibody preparation procedures, such as those described, for example,in Harlow et al. (1988, In: Antibodies, A Laboratory Manual, Cold SpringHarbor, N.Y.) and in Tuszynski et al. (1988, Blood, 72:109-115).Quantities of the desired peptide may also be synthesized using chemicalsynthesis technology. Alternatively, DNA encoding the desired peptidemay be cloned and expressed from an appropriate promoter sequence incells suitable for the generation of large quantities of peptide.Monoclonal antibodies directed against the peptide are generated frommice immunized with the peptide using standard procedures as referencedherein.

Nucleic acid encoding the monoclonal antibody obtained using theprocedures described herein may be cloned and sequenced using technologywhich is available in the art, and is described, for example, in Wrightet al. (1992, Critical Rev. Immunol. 12:125-168), and the referencescited therein. Further, the antibody of the invention may be “humanized”using the technology described in, for example, Wright et al., and inthe references cited therein, and in Gu et al. (1997, Thrombosis andHematocyst 77:755-759), and other methods of humanizing antibodieswell-known in the art or to be developed.

In one embodiment of the invention, a phage antibody library may begenerated, as described in detail elsewhere herein. To generate a phageantibody library, a cDNA library is first obtained from mRNA which isisolated from cells, e.g., peripheral blood lymphocytes, which expressthe desired protein to be expressed on the phage surface, e.g., thedesired antibody. cDNA copies of the mRNA are produced using reversetranscriptase. cDNA which specifies immunoglobulin fragments areobtained by PCR and the resulting DNA is cloned into a suitablebacteriophage vector to generate a bacteriophage DNA library comprisingDNA specifying immunoglobulin genes. The procedures for making abacteriophage library comprising heterologous DNA are well known in theart and are described, for example, in Sambrook et al., supra.

Bacteriophage which encode the desired antibody, may be engineered suchthat the protein is displayed on the surface thereof in such a mannerthat it is available for binding to its corresponding binding peptide,e.g., the antigen against which the antibody is directed, such as anantigen of interest (i.e. the PEDF peptide fragments of the invention).Thus, when bacteriophage which express a specific antibody are incubatedin the presence of the corresponding antigen, the bacteriophage willbind to the antigen. Bacteriophage which do not express the antibodywill not bind to the antigen. Such panning techniques are well known inthe art and are described for example, in Wright et al. (supra).

Processes such as those described above, have been developed for theproduction of human antibodies using M13 bacteriophage display (Burtonet al., 1994, Adv. Immunol. 57:191-280). Essentially, a cDNA library isgenerated from mRNA obtained from a population of antibody-producingcells. The mRNA encodes rearranged immunoglobulin genes and thus, thecDNA encodes the same. Amplified cDNA is cloned into M13 expressionvectors creating a library of phage which express human Fab fragments ontheir surface. Phage which display the antibody of interest are selectedby antigen binding and are propagated in bacteria to produce solublehuman Fab immunoglobulin. Thus, in contrast to conventional monoclonalantibody synthesis, this procedure immortalizes DNA encoding humanimmunoglobulin rather than cells which express human immunoglobulin.

The procedures just presented describe the generation of phage whichencode the Fab portion of an antibody molecule. However, the inventionshould not be construed to be limited solely to the generation of phageencoding Fab antibodies. Rather, phage which encode single chainantibodies (scFv/phage antibody libraries) are also included in theinvention. Fab molecules comprise the entire Ig light chain, that is,they comprise both the variable and constant region of the light chain,but include only the variable region and first constant region domain(CH1) of the heavy chain. Single chain antibody molecules comprise asingle chain of protein comprising the Ig Fv fragment. An Ig Fv fragmentincludes only the variable regions of the heavy and light chains of theantibody, having no constant region contained therein. Phage librariescomprising scFv DNA may be generated following the procedures describedin Marks et al. (1991, J. Mol. Biol. 222:581-597). Panning of phage sogenerated for the isolation of a desired antibody is conducted in amanner similar to that described for phage libraries comprising Fab DNA.

The invention should also be construed to include synthetic phagedisplay libraries in which the heavy and light chain variable regionsmay be synthesized such that they include nearly all possiblespecificities (Barbas, 1995, Nature Medicine 1:837-839;

de Kruif et al. 1995, J. Mol. Biol. 248:97-105). In another embodimentof the invention, phage-cloned antibodies derived from immunized animalscan be humanized by known techniques.

Stem Cells

The peptides of the present invention are useful as a niche factor formaintaining stem cell populations. In certain embodiments, the presentinvention comprises a method of maintaining a stem cell comprisingcontacting the stem cell with one or more peptides disclosed herein. Forexample, the method may comprise administering the one or more peptidesor administering one or more isolated nucleic acids encoding the one ormore peptides. In certain embodiments, the method comprises contactingthe stem cell with a culture medium, buffer, or the like comprising oneor more peptides. In one embodiment, the method comprises geneticallymodifying a cell to express, and in certain instances secrete, one ormore of the peptides disclosed herein by contacting the cell with one ormore isolated nucleic acids encoding the one or more peptides. Incertain embodiments, the genetically modified cell may be the stem cellor another cell. For example, in one embodiment, the geneticallymodified cell may be a cell co-cultured with the stem cell.

The method may be used to maintain one or more of any type of stem cell,including, but not limited to, pluripotent stem cell, multipotent stemcells, embryonic stem cells, somatic stem cells, cord blood-derived stemcells, induced pluripotent stem cells, hematopoietic stem cells,mesenchymal stem cells, neural stem cells, and the like.

In certain embodiments, the method comprises culturing a stem cell in asuitable culture medium, wherein the culture medium is supplemented withone or more peptides of the present invention.

In one embodiment, the stem cell is genetically modified to express, andin certain instances secrete, one or more of the peptides describedherein.

In certain embodiments, the presence of one or more peptides of thepresent invention in a stem cell niche or in a culture medium promotesthe self-renewal, differentiation, de-differentiation, proliferation,and/or expansion of a stem cell or stem cell population.

Therapeutic Methods

The peptides disclosed herein are useful in any therapeutic orprophylactic treatment known in the art, or subsequently discovered, forPEDF protein. Such therapeutic applications are disclosed in detail in,for instance, U.S. Pat. Nos. 6,451,763; 6,288,024; 6,391,850; 6,670,333;6,797,691; 6,919,309 and US Patent Publication 20060008900, each ofwhich is enclosed herein in its entirety. Methods of treatment may betherapeutic or prophylactic.

In one embodiment, the peptides of the invention are useful in treatinga variety of pathologies including but not limited to inflammation, celldeath, vascular leakage/neovascularization, and the like.

The peptides of the invention may be administered to a treatment siteusing any method known in the art. Exemplary pharmaceutical compositionsand routes of administration are detailed elsewhere herein. In certainembodiments, the administration of one or more peptides of the inventioncomprises administering one or more peptides along with a drug deliveryvehicle. The drug delivery vehicle may be a microparticle, nanoparticle,liposome, micelle, or other vehicle known in the art. The drug deliveryvehicle may be administered locally into one or more regions of the eyeor systemically.

In certain embodiments, administration of one or more peptides comprisesadministration of a cell comprising the one or more peptides or anucleic acid encoding the one or more peptides. For example, in oneembodiment, the method comprises administering to a subject a stem cellcomprising a nucleic acid encoding one or more stem cells of theinvention.

In certain embodiments, the method comprises a gene therapy method e.g.,administration of an isolated nucleic acid encoding a peptide of thepresent invention. Engineering of such isolated nucleic acids byrecombinant DNA or RNA methods is well within the ability of one skilledin the art. Codon optimization, for purposes of maximizing recombinantprotein yields in particular cell backgrounds, is also well within theability of one skilled in the art. Administration of an isolated nucleicacid encoding the peptide is encompassed by the expression“administering a therapeutically effective amount of a peptide of theinvention”. Gene therapy methods are well known in the art. See, e.g.,WO96/07321 which discloses the use of gene therapy methods to generateintracellular antibodies. Gene therapy methods have also beensuccessfully demonstrated in human patients. See, e.g., Baumgartner etal., Circulation 97: 12, 1114-1123 (1998), and more recently, Fatham,C.G. ‘A gene therapy approach to treatment of autoimmune diseases’,Immun. Res. 18:15-26 (2007); and U.S. Pat. No. 7,378089, bothincorporated herein by reference. See also Bainbridge JWB et al. “Effectof gene therapy on visual function in Leber's congenital Amaurosis”. NEngl J Med 358:2231-2239, 2008; and Maguire A M et al. “Safety andefficacy of gene transfer for Leber's Congenital Amaurosis”. N Engl JMed 358:2240-8, 2008.

There are two major approaches for introducing a nucleic acid encodingthe peptide (optionally contained in a vector) into a patients cells; invivo and ex vivo. For in vivo delivery the nucleic acid is injecteddirectly into the patient, usually at the site where the peptide isrequired. For ex vivo treatment, the patient's cells are removed, thenucleic acid is introduced into these isolated cells and the modifiedcells are administered to the patient either directly or, for example,encapsulated within porous membranes which are implanted into thepatient (see, e.g., U.S. Pat. Nos. 4,892,538 and 5,283,187). There are avariety of techniques available for introducing nucleic acids intoviable cells. The techniques vary depending upon whether the nucleicacid is transferred into cultured cells in vitro, or in vivo in thecells of the intended host. Techniques suitable for the transfer ofnucleic acid into mammalian cells in vitro include the use of liposomes,electroporation, microinjection, cell fusion, DEAE-dextran, the calciumphosphate precipitation method, etc. Commonly used vectors for ex vivodelivery of the gene are retroviral and lentiviral vectors.

Preferred in vivo nucleic acid transfer techniques include transfectionwith viral vectors such as adenovirus, Herpes simplex I virus,adeno-associated virus), lipid-based systems (useful lipids forlipid-mediated transfer of the gene are DOTMA, DOPE and DC-Chol, forexample), naked DNA, and transposon-based expression systems. For reviewof the currently known gene marking and gene therapy protocols seeAnderson et al., Science 256:808-813 (1992). See also WO 93/25673 andthe references cited therein.

“Gene therapy” includes both conventional gene therapy where a lastingeffect is achieved by a single treatment, and the administration of genetherapeutic agents, which involves the one time or repeatedadministration of a therapeutically effective DNA or mRNA.Oligonucleotides can be modified to enhance their uptake, e.g. bysubstituting their negatively charged phosphodiester groups by unchargedgroups. Peptides of the present invention can be delivered using genetherapy methods, for example locally in a region of the eye, orsystemically (e.g., via vectors that selectively target specific tissuetypes, for example, tissue-specific adeno-associated viral vectors). Insome embodiments, primary cells (such as lymphocytes or stem cells) fromthe individual can be transfected ex vivo with a gene encoding any ofthe peptides of the present invention, and then returning thetransfected cells to the individual's body.

Diabetic Retinopathy

Over 350 million individuals world-wide suffer from diabetes with hikingrates expected in the next 10 years due to the current obesity pandemic.As many as 10% of these individuals suffer from mild to severe loss ofvision in a condition known as diabetic retinopathy which is caused bydamage to neurons in the retina and hemorrhaging in the eye. Currentlythey are not effective treatments for diabetic retinopathy and there isan urgent need to develop biotherapeutics. Laser surgery is used insevere cases to reduce hemorrhaging but often results in more damage tovisual neurons. Drugs currently under investigation for diabeticretinopathy are of the “anti-VEGF” class including Lucentis, Avastin,and the VEGF Trap, which are too large to deliver as eye drops and havelimited efficacy. These are costly (>$1000/treatment), must be injectedintraocularly −8-12 times/year, and only reduce vascular pathology. Theydo not prevent damage to visual neurons, which degenerate long beforevascular leakage is detected in diabetic retinopathy. While controllingvascular leakage can prevent partial loss of vision, diabeticretinopathy begins in neurons that are degenerating much earlier in theretina.

The peptides of the present invention may be used in methods of treatingdiabetic retinopathy. In particular, a therapeutic amount of a peptideof the present invention is administered to a patient with diabeticretinopathy. Without being held to any particular theory, it is believedthat the peptides of the present invention are able to help treat avariety of the pathological conditions relating to diabetic retinopathyincluding inflammation, cell death vascular leakage and macular edema.In certain embodiments, the peptides of the invention target threehallmark pathologies of diabetic retinopathy-inflammation, cell death,and vascular leakage. There is currently no known product that cantarget all three features of diabetic retinopathy. While any of thepeptides of the present invention may be used to treat diabeticretinopathy and the related pathologies of diabeticretinopathy(including inflammation, cell death ,vascular leakage andmacular edema) in preferred embodiments the peptide is SpxA1, 81-2,81-5, 81-12, 81-13 or 81-20 or combinations of these peptides. Thepeptide can be administered using any techniques known in the art and ina preferred embodiment the peptide is administered in the form of an eyedrop.

Retinopathy of Prematurity

Retinopathy of prematurity (ROP) is a blinding disease seen in children.ROP has two phases. The first phase begins with delayed retinal vasculargrowth after birth and partial regression of existing vessels. Thesecond phase is associated with hypoxia-induced pathological vesselgrowth. It is thought that excessive oxygen contributes to ROP throughregulation of vascular endothelial growth factor (VEGF). Suppression ofVEGF by oxygen in phase I of ROP inhibits normal vessel growth, whereaselevated levels of VEGF induced by hypoxia in phase II of ROPprecipitate pathological vessel proliferation

The peptides of the present invention may be used in methods of treatingROP in particular a therapeutic amount of a peptide of the presentinvention is administered to the patient with ROP. In certainembodiments, the peptides of the invention reduce the levels of VEGF inthe retina, a key factor in the growth of endothelial cells and bloodvessels. While any of the peptides of the present invention may be used,in preferred embodiments, the peptide is SpxA1, 81-2, 81-5, 81-12, 81-13or 81-20 or combinations of these peptides. The peptide can beadministered using any techniques known in the art and in a preferredembodiment the peptide is administered in the form of an eye drop.

Age-Related Macular Degeneration

Individuals over the age of forty are susceptible to Age-Related MacularDegeneration (“AMD”), a condition where neuronal cells of the retina dieand in the wet form of the disease, neovasularization is prominent. AMDaffects more than 9.1 million individuals in the USA (2010 data).Current Anti-VEGF drugs used to treat AMD are only initially effectivein about 70% of all patients with the wet form of the disease, withdecreasing efficacy over time, and there are no treatments for the dryform of AMD.

The peptides of the present invention may be used in methods of treatingAMD in particular a therapeutic amount of a peptide of the presentinvention is administered to the patient with AMD. In certainembodiments, as in diabetic retinopathy, the peptides of the inventionreduce vascular leakage, inflammation, and cell death—which, likediabetic retinopathy, are predominant features in. While any of thepeptides of the present invention may be used, in preferred embodiments,the peptide is SpxA1, 81-2, 81-5, 81-10, 81-12, 81-13, 81-19 or 81-20 orcombinations of these peptides. The peptide can be administered usingany techniques known in the art and in a preferred embodiment thepeptide is administered in the form of an eye drop.

Retinitis Pigmentosa

Retinitis pigmentosa is a group of inherited disorders in whichabnormalities of the photoreceptors (rods or cones) or the retinalpigment epithelium of the retina lead to progressive visual loss. Someforms of retinitis pigmentosa are dominant, requiring only one gene fromeither parent; others are X-linked, requiring only one gene from themother. In some people, mostly males, an inherited form of hearing lossalso develops.

The retinal pigment epithelium provides nutrients and support to thephotoreceptor cells of the retina, in particular, inhibitors ofoxidative stress and apoptosis. For example, neurotrophins of theretinal pigment epithelium activate the release of anti-inflammatory andanti-oxidative factors.

In retinitis pigmentosa, there is chronic death of photoreceptor cells(rods and cones) of the retina. These photoreceptor cells, which areresponsible for vision when light is low, gradually degenerate, so thatvision becomes poor in the dark. The first symptoms of retinitispigmentosa often begin in early childhood. Over time, a progressive lossof peripheral vision occurs. In the late stages of the disease, a personhas a small area of central vision and a little peripheral visionremaining (tunnel vision). A need exists in the art for methods oftreating retinitis pigmentosa.

The peptides of the present invention may be used in methods of treatretinitis pigmentosa. In particular a therapeutic amount of a peptide ofthe present invention is administered to the patient with retinitispigmentosa. In certain embodiments, the neuroprotective action of thepeptides of the invention reduces neuronal cell death and thus delays orprevents vision loss in the disease. While any of the peptides of thepresent invention may be used, in preferred embodiments, the peptide isSpxA1, 81-2, 81-5, 81-12, 81-13 or 81-20 or combinations of thesepeptides. The peptide can be administered using any techniques known inthe art and in a preferred embodiment the peptide is administered in theform of an eye drop.

Glaucoma

Glaucoma is one of the three leading causes of blindness in the UnitedStates and it is a leading cause of blindness in the world. Over 2.2million people in the United States have glaucoma, and several millionmore are at risk of developing the disease. As the population ages, thenumber of individuals with glaucoma will continue to grow since glaucomaaffects the oldest individuals disproportionately. Glaucoma is not justone disease, rather, it is a spectrum of conditions that share a finalcommon pathway of acquired, progressive deterioration of the neuronalcomponents of the optic nerve. Neuronal death results in loss of visiononce a sufficient number of individual nerves are destroyed.

Factors associated with the development of glaucoma and its progressionhave been identified and are in the process of being clarified. Elevatedintraocular pressure (IOP) is the leading cause of glaucoma. Pressure iselevated because drainage of aqueous fluid from within the eye isimpaired. Current treatments for glaucoma center on reducing pressure inthe eye by reducing the amount of aqueous fluid being produced or byenhancing the flow of fluid out of the eye by mechanical or other means.Currently available drugs do not enhance or restore functioning of thenatural drainage pathway.

Glaucoma patients may also suffer reduced blood flow to the optic nerveand neuronal tissue, diminished resistance of the nerve tissue todamage, and compliance of connective tissue surrounding and supportingthe optic nerve. Current treatments do not address any such factors.

The peptides of the present invention may be used in methods of treatglaucoma in particular a therapeutic amount of a peptide of the presentinvention is administered to the patient with glaucoma. Much of the celldeath seen in glaucoma is believed to be the result of oxidative stress.In certain embodiments, the peptides of the invention protects againstoxidative stress and are thus reduces the ganglion cell loss. While anyof the peptides of the present invention may be used, in preferredembodiments the peptide is SpxA1, 81-2, 81-5, 81-12, 81-13 or 81-20 orcombinations of these peptides. The peptide can be administered usingany techniques known in the art and in a preferred embodiment thepeptide is administered in the form of an eye drop.

Uveitis

Uveitis refers to inflammation of the uvea, which is the middle,pigmented vascular structures of the eye, including the iris ciliarybody and choroid. While uveitis is usually an isolated disorder, it issometimes associated with one or more systemic or ophthalmic conditions.In some instances uveitis has infectious causes, where the presence ofan infection results in an immune response which causes the inflammationof the uvea. In other instances, uveitis is caused by an autoimmuneresponse, sometimes associated with systemic autoimmune disorders. Thus,increased levels of inflammatory markers are often observed both in theeye as well as in the serum of affected subjects.

Any of the peptides of the present invention may be used to treatuveitis. The peptide can be administered using any techniques known inthe art and in a preferred embodiment the peptide is administered in theform of an eye drop.

Corneal Inflammation and Angiogenesis

Inflammation of the cornea may have numerous causes, includingauto-immune mediated inflammation, infectious keratitis, or a heightenedimmune response to non-pathologic disturbances. Infectious keratitis maybe caused by bacterial, fungal, viral, or parasitic invasion. If nottreated adequately, corneal inflammation may lead to fibrinizationsecondary to inflammatory cells, granulomatous formation, deposition offibroblasts, tissue hardening and destruction, neovascularization andpannus.

Any of the peptides of the present invention may be used to treatuveitis. The peptide can be administered using any techniques known inthe art and in a preferred embodiment the peptide is administered in theform of an eye drop.

Other Diseases

The peptides of the invention can be used to treat a variety of diseasesand disorders. In one embodiment, the peptides of the invention areuseful in treating a variety of pathologies including but not limited toinflammation, cell death, vascular leakage/neovascularization, and thelike.

Diabetes is associated with a high incidence of complications includingretinopathy, nephropathy and peripheral neuropathy. It is also a majorrisk factor for cardiovascular diseases including stroke, and coronaryartery disease. Diabetes is known to be associated with a state ofgeneral chronic, low-level inflammation, which precedes, and is believedto promote, insulin resistance and increase risk for cardiovasculardisease. Obesity and other metabolic syndrome disorders that frequentlylead to diabetes, including arterial hypertension, insulin resistance,dyslipidemia, and abdominal obesity, are also linked to chronicinflammatory response caused by elevated levels of proinflammatorycytokines such as TNFα. As well as inflammation these diseases areassociated with abnormal regulation of VEGF, neovascularization andvascular leakage. For example, in diabetic nephropathy there areincreased levels of VEGF and angiogenesis as well as increasedinflammatory cytokines including TNFα.

The compositions of the invention reduce levels of the proangiogenic andvascular leakage factor VEGF, and the inflammatory molecules, TNFα, andIFNγ. Such actions make these peptides important agents in themanagement of a wide range of diabetic complications and metabolicdiseases where chronic inflammation and dysregulation of VEGF areconsidered key factors in the disease onset and progression.

The peptides disclosed herein may be used to treat or prevent diseasesor disorders involving neuronal degeneration, including, but not limitedto, nerve injuries, neurodegenerative diseases, and ocular diseases anddisorders, including, but not limited to, transient or chronic ischemicinjury. In particular, nerve injuries and neurodegenerative diseases ofneurons in the retina, brain and spinal cord are beneficially treatedusing a peptide of the invention. Non-limiting examples of such diseasesand disorders include Parkinson's disease, Huntington's disease,Alzheimer's disease, amyotrophic lateral sclerosis, multiple sclerosis,diabetic retinopathy, retinopathy of prematurity, macular degeneration,glaucoma, venous stasis retinopathy, ischemic oculopathy and ocularischemic syndrome.

Neural transplantation can be used to treat nerve injuries andneurodegenerative diseases. Treatment of neuronal cells withcompositions of the invention to enhance nerve cell survival is alsoembraced by the invention. Treatment may occur before, during or afterneural transplantation. Similarly, transfection of either neurons orastroglia with an expression vector comprising a coding sequence for apeptide of the invention before implantation can provide a long-termsource of a peptide at the transplantation site. Transplantation ofneural retina and photoreceptor cells is contemplated to benefit fromthe treatment with a composition of the invention of the inventionbefore, during or after transplantation. Alternatively, an expressionvector comprising a coding sequence for a peptide of the invention canbe transfected at high levels into adjacent retinal pigment epithelial(RPE) cells where they can serve as a source of the peptide.

In one embodiment, the peptides of the invention are useful in treatinga condition including but is not limited sepsis, acute respiratorydistress syndrome, nephrotic syndrome, diabetic neuropathy,preproliferative diabetic retinopathy, cancer, or proliferative diabeticretinopathy.

In one embodiment, the peptides of the invention can inhibit the growthof blood vessels within and to the tumor, and in some cases, inducetumor cells to differentiate and thus divide slowly. Inhibiting thegrowth of blood vessels within tumors prevents sufficient nutrients andoxygen from being supplied to the tumor to support growth beyond a givensize. Thus, the inventive method can prevent the nucleation of tumorsfrom cancerous cells already present due to genetic predisposition orthe presence of external carcinogens (e.g., tobacco, alcohol, industrialsolvents, etc.). Aside from preventing tumorigenesis, the inventivemethod can retard the growth of existing tumors, thus rendering themmore easily contained and excised and may cause them to regress. Thisapplication is highly advantageous for treating tumors that aredifficult to operate on (e.g., brain or prostate tumors). In addition,the method is useful for treatment of childhood tumors, including, butnot limited to, neuroblastoma. Moreover, minimizing the number of bloodvessels within existing tumors lessens the probability that the tumorwill metastasize. In treating tumors, the method can be used alone or inconjunction with other treatments, to control the growth of tumors.Indeed, employing the inventive method can potentiate the response ofsome tumors to other therapies. For example, the inventive methodoptionally can be employed as a pretreatment for (e.g., for about a weekin advance of), and continued during, a chemotherapeutic or radiationregimen. The method of the invention may also be used in conjunctionwith the use of biological response modifiers, such as for example,interferon, or other anti-angiogenic agents, and also is useful inconjunction with the use of agents which induce the production ofanti-angiogenic agents in vivo. Further, the method of the invention maybe used in conjunction with agents which promote the differentiation ofcells, particularly, but not limited to agents which promote thedifferentiation of brain tumor cells.

In one embodiment, the peptides of the invention are useful for theprevention of neovascularization. Thus, for example, the inventivemethod can be used as part of a treatment for disorders of blood vessels(e.g., hemangiomas and capillary proliferation within atheroscleroticplaques), muscle (e.g., myocardial angiogenesis or angiogenesis withinsmooth muscles), joints (e.g., arthritis, hemophiliac joints, etc.), andother disorders associated with angiogenesis (e.g., Osler-WebberSyndrome, plaque neovascularization, telangiectasia, angiofibroma, woundgranularization, etc.). In addition, the invention is useful fortreatment of nasal polyps, especially in cystic fibrosis patients,leukemia which stems from bone marrow cell abnormal growth, and prostatecancer. The invention can be construed in general to be useful fortreatment of benign neoplasias.

The inventive method is also useful as a means of preventing theoccurrence of a disease or disorder associated with vascularpermeability or angiogenesis, i.e., the methods are useful asprophylactic methods for the prevention of disease in patients at riskfor the disease. For example, and without limitation, the peptides ofthe invention can be used to prevent the onset of diabetic retinopathyin a patient having diabetes, to prevent the onset of cancer in personsknown to be at risk for certain cancers, and the like. Thus, the methodsof the invention should not be construed as being limited to treatmentof overt disease, but rather, should be construed as being useful forthe prevention of disease in patients who are at risk.

The invention should also be construed to include treatment ofprecancerous lesions, for example, but without limitation, nasal polyps,particularly in patients having cystic fibrosis. Nasal polyps in thesepatients are angiogenic, and further, the cerebral spinal fluid ofcystic fibrosis patients contains an excess of the angiogenic factorVEGF. Alleviation of these conditions, especially in cystic fibrosispatients, wherein the alleviation comprises administration of thepeptides of the invention is therefore included in the presentinvention.

Within the context of the inventive method, peptides of the inventioncan be supplied alone or in conjunction with other known antiangiogenicfactors. For example, the peptides of the invention can be used inconjunction with antibodies and peptides that block integrin engagement,proteins and small molecules that inhibit metalloproteinases (e.g.,marmistat), agents that block phosphorylation cascades withinendothelial cells (e.g., herbamycin), dominant negative receptors forknown inducers of angiogenesis, antibodies against inducers ofangiogenesis or other compounds that block their activity (e.g.,suramin), or other compounds (e.g., retinoids, IL-4, interferons, etc.)acting by other means. Indeed, as such factors modulate angiogenesis bydifferent mechanisms, employing peptides of the invention in combinationwith other antiangiogenic agents can potentiate a more potent (andpotentially synergistic) inhibition of angiogenesis within the desiredtissue.

Pharmaceutical Compositions and Formulations

The invention also encompasses the use of pharmaceutical compositions ofthe invention or salts thereof to practice the methods of the invention.Such a pharmaceutical composition may consist of at least one compoundor conjugate of the invention or a salt thereof in a form suitable foradministration to a subject, or the pharmaceutical composition maycomprise at least one compound or conjugate of the invention or a saltthereof, and one or more pharmaceutically acceptable carriers, one ormore additional ingredients, or some combination of these. The compoundor conjugate of the invention may be present in the pharmaceuticalcomposition in the form of a physiologically acceptable salt, such as incombination with a physiologically acceptable cation or anion, as iswell known in the art.

The peptides of the invention may be administered by a variety ofmethods including administration as an eye drop, administration byintraocular injection, administration as a gel to the eye,administration as an implant in the eye that releases the peptide overtime, administration as a virus that expresses the peptide, andadministration using a cell-based expression system. Each of thesemethods are known in the art and are discussed in more detail below.

In an embodiment, the pharmaceutical compositions useful for practicingthe methods of the invention may be administered to deliver a dose ofbetween 1 ng/kg/day and 100 mg/kg/day. In another embodiment, thepharmaceutical compositions useful for practicing the invention may beadministered to deliver a dose of between 1 ng/kg/day and 500 mg/kg/day.

The relative amounts of the active ingredient, the pharmaceuticallyacceptable carrier, and any additional ingredients in a pharmaceuticalcomposition of the invention will vary, depending upon the identity,size, and condition of the subject treated and further depending uponthe route by which the composition is to be administered. By way ofexample, the composition may comprise between 0.1% and 100% (w/w) activeingredient.

Pharmaceutical compositions that are useful in the methods of theinvention may be suitably developed for oral, rectal, vaginal,parenteral, topical, pulmonary, intranasal, buccal, ophthalmic, oranother route of administration. A composition useful within the methodsof the invention may be directly administered to the skin, vagina or anyother tissue of a mammal. Other contemplated formulations includeliposomal preparations, resealed erythrocytes containing the activeingredient, and immunologically-based formulations. The route(s) ofadministration will be readily apparent to the skilled artisan and willdepend upon any number of factors including the type and severity of thedisease being treated, the type and age of the veterinary or humansubject being treated, and the like.

The formulations of the pharmaceutical compositions described herein maybe prepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with a carrier or one ormore other accessory ingredients, and then, if necessary or desirable,shaping or packaging the product into a desired single- or multi-doseunit.

As used herein, a “unit dose” is a discrete amount of the pharmaceuticalcomposition comprising a predetermined amount of the active ingredient.The amount of the active ingredient is generally equal to the dosage ofthe active ingredient that would be administered to a subject or aconvenient fraction of such a dosage such as, for example, one-half orone-third of such a dosage. The unit dosage form may be for a singledaily dose or one of multiple daily doses (e.g., about 1 to 4 or moretimes per day). When multiple daily doses are used, the unit dosage formmay be the same or different for each dose.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions that aresuitable for ethical administration to humans, it will be understood bythe skilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist maydesign and perform such modification with merely ordinary, if any,experimentation. Subjects to which administration of the pharmaceuticalcompositions of the invention is contemplated include, but are notlimited to, humans and other primates, mammals including commerciallyrelevant mammals such as cattle, pigs, horses, sheep, cats, and dogs.

In one embodiment, the compositions of the invention are formulatedusing one or more pharmaceutically acceptable excipients or carriers. Inone embodiment, the pharmaceutical compositions of the inventioncomprise a therapeutically effective amount of a compound or conjugateof the invention and a pharmaceutically acceptable carrier.Pharmaceutically acceptable carriers that are useful, include, but arenot limited to, glycerol, water, saline, ethanol and otherpharmaceutically acceptable salt solutions such as phosphates and saltsof organic acids. Examples of these and other pharmaceuticallyacceptable carriers are described in Remington's Pharmaceutical Sciences(1991, Mack Publication Co., New Jersey).

The carrier may be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity may be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms may be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol,in the composition. Prolonged absorption of the injectable compositionsmay be brought about by including in the composition an agent thatdelays absorption, for example, aluminum monostearate or gelatin. In oneembodiment, the pharmaceutically acceptable carrier is not DMSO alone.

Formulations may be employed in admixtures with conventional excipients,i.e., pharmaceutically acceptable organic or inorganic carriersubstances suitable for oral, vaginal, parenteral, nasal, intravenous,subcutaneous, ophthalmic, enteral, or any other suitable mode ofadministration, known to the art. The pharmaceutical preparations may besterilized and if desired mixed with auxiliary agents, e.g., lubricants,preservatives, stabilizers, wetting agents, emulsifiers, salts forinfluencing osmotic pressure buffers, coloring, flavoring and/oraromatic substances and the like. They may also be combined wheredesired with other active agents, e.g., other analgesic agents.

As used herein, “additional ingredients” include, but are not limitedto, one or more of the following: excipients; surface active agents;dispersing agents; inert diluents; granulating and disintegratingagents; binding agents; lubricating agents; sweetening agents; flavoringagents; coloring agents; preservatives; physiologically degradablecompositions such as gelatin; aqueous vehicles and solvents; oilyvehicles and solvents; suspending agents; dispersing or wetting agents;emulsifying agents, demulcents; buffers; salts; thickening agents;fillers; emulsifying agents; antioxidants; antibiotics; antifungalagents; stabilizing agents; and pharmaceutically acceptable polymeric orhydrophobic materials. Other “additional ingredients” that may beincluded in the pharmaceutical compositions of the invention are knownin the art and described, for example in Genaro, ed. (1985, Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa.), which isincorporated herein by reference.

The composition of the invention may comprise a preservative from about0.005% to 2.0% by total weight of the composition. The preservative isused to prevent spoilage in the case of exposure to contaminants in theenvironment. Examples of preservatives useful in accordance with theinvention included but are not limited to those selected from the groupconsisting of benzyl alcohol, sorbic acid, parabens, imidurea andcombinations thereof. A particularly preferred preservative is acombination of about 0.5% to 2.0% benzyl alcohol and 0.05% to 0.5%sorbic acid.

Liquid suspensions may be prepared using conventional methods to achievesuspension of the active ingredient in an aqueous or oily vehicle.Aqueous vehicles include, for example, water, and isotonic saline. Oilyvehicles include, for example, almond oil, oily esters, ethyl alcohol,vegetable oils such as arachis, olive, sesame, or coconut oil,fractionated vegetable oils, and mineral oils such as liquid paraffin.Liquid suspensions may further comprise one or more additionalingredients including, but not limited to, suspending agents, dispersingor wetting agents, emulsifying agents, demulcents, preservatives,buffers, salts, flavorings, coloring agents, and sweetening agents. Oilysuspensions may further comprise a thickening agent. Known suspendingagents include, but are not limited to, sorbitol syrup, hydrogenatededible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gumacacia, and cellulose derivatives such as sodium carboxymethylcellulose,methylcellulose, hydroxypropylmethylcellulose. Known dispersing orwetting agents include, but are not limited to, naturally-occurringphosphatides such as lecithin, condensation products of an alkyleneoxide with a fatty acid, with a long chain aliphatic alcohol, with apartial ester derived from a fatty acid and a hexitol, or with a partialester derived from a fatty acid and a hexitol anhydride (e.g.,polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylenesorbitol monooleate, and polyoxyethylene sorbitan monooleate,respectively). Known emulsifying agents include, but are not limited to,lecithin, and acacia. Known preservatives include, but are not limitedto, methyl, ethyl, or n-propyl-para-hydroxybenzoates, ascorbic acid, andsorbic acid. Known sweetening agents include, for example, glycerol,propylene glycol, sorbitol, sucrose, and saccharin. Known thickeningagents for oily suspensions include, for example, beeswax, hardparaffin, and cetyl alcohol.

Liquid solutions of the active ingredient in aqueous or oily solventsmay be prepared in substantially the same manner as liquid suspensions,the primary difference being that the active ingredient is dissolved,rather than suspended in the solvent. As used herein, an “oily” liquidis one which comprises a carbon-containing liquid molecule and whichexhibits a less polar character than water. Liquid solutions of thepharmaceutical composition of the invention may comprise each of thecomponents described with regard to liquid suspensions, it beingunderstood that suspending agents will not necessarily aid dissolutionof the active ingredient in the solvent. Aqueous solvents include, forexample, water, and isotonic saline. Oily solvents include, for example,almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis,olive, sesame, or coconut oil, fractionated vegetable oils, and mineraloils such as liquid paraffin.

Powdered and granular formulations of a pharmaceutical preparation ofthe invention may be prepared using known methods. Such formulations maybe administered directly to a subject, used, for example, to formtablets, to fill capsules, or to prepare an aqueous or oily suspensionor solution by addition of an aqueous or oily vehicle thereto. Each ofthese formulations may further comprise one or more of dispersing orwetting agent, a suspending agent, and a preservative. Additionalexcipients, such as fillers and sweetening, flavoring, or coloringagents, may also be included in these formulations.

A pharmaceutical composition of the invention may also be prepared,packaged, or sold in the form of oil-in-water emulsion or a water-in-oilemulsion. The oily phase may be a vegetable oil such as olive or arachisoil, a mineral oil such as liquid paraffin, or a combination of these.Such compositions may further comprise one or more emulsifying agentssuch as naturally occurring gums such as gum acacia or gum tragacanth,naturally-occurring phosphatides such as soybean or lecithinphosphatide, esters or partial esters derived from combinations of fattyacids and hexitol anhydrides such as sorbitan monooleate, andcondensation products of such partial esters with ethylene oxide such aspolyoxyethylene sorbitan monooleate. These emulsions may also containadditional ingredients including, for example, sweetening or flavoringagents.

Methods for impregnating or coating a material with a chemicalcomposition are known in the art, and include, but are not limited tomethods of depositing or binding a chemical composition onto a surface,methods of incorporating a chemical composition into the structure of amaterial during the synthesis of the material (i.e., such as with aphysiologically degradable material), and methods of absorbing anaqueous or oily solution or suspension into an absorbent material, withor without subsequent drying.

Administration/Dosing

The regimen of administration may affect what constitutes an effectiveamount of a therapeutic composition of the invention. The therapeuticformulations may be administered to the subject either prior to or aftera diagnosis of disease. Further, several divided dosages, as well asstaggered dosages may be administered daily or sequentially, or the dosemay be continuously infused, or may be a bolus injection. Further, thedosages of the therapeutic formulations may be proportionally increasedor decreased as indicated by the exigencies of the therapeutic orprophylactic situation.

In certain embodiments, administration of the composition of theinvention is non-invasive. For example, in certain embodimentsadministration comprises ophthalmic delivery, for example by way of aneye drop or other ophthalmic formulation.

Administration of the compositions of the present invention to asubject, preferably a mammal, more preferably a human, may be carriedout using known procedures, at dosages and for periods of time effectiveto prevent or treat disease. An effective amount of the therapeuticcompound necessary to achieve a therapeutic effect may vary according tofactors such as the activity of the particular compound employed; thetime of administration; the rate of excretion of the compound; theduration of the treatment; other drugs, compounds or materials used incombination with the compound; the state of the disease or disorder,age, sex, weight, condition, general health and prior medical history ofthe subject being treated, and like factors well-known in the medicalarts. Dosage regimens may be adjusted to provide the optimum therapeuticresponse. For example, several divided doses may be administered dailyor the dose may be proportionally reduced as indicated by the exigenciesof the therapeutic situation. A non-limiting example of an effectivedose range for a therapeutic compound of the invention is from about 1and 5,000 mg/kg of body weight/per day. One of ordinary skill in the artwould be able to study the relevant factors and make the determinationregarding the effective amount of the therapeutic compound without undueexperimentation.

The compound may be administered to a subject as frequently as severaltimes daily, or it may be administered less frequently, such as once aday, once a week, once every two weeks, once a month, or even lessfrequently, such as once every several months or even once a year orless. It is understood that the amount of compound dosed per day may beadministered, in non-limiting examples, every day, every other day,every 2 days, every 3 days, every 4 days, or every 5 days. For example,with every other day administration, a 5 mg per day dose may beinitiated on Monday with a first subsequent 5 mg per day doseadministered on Wednesday, a second subsequent 5 mg per day doseadministered on Friday, and so on. The frequency of the dose will bereadily apparent to the skilled artisan and will depend upon any numberof factors, such as, but not limited to, the type and severity of thedisease being treated, and the type and age of the animal.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient that is effective to achieve the desiredtherapeutic response for a particular subject, composition, and mode ofadministration, without being toxic to the subject.

A medical doctor, e.g., physician or veterinarian, having ordinary skillin the art may readily determine and prescribe the effective amount ofthe pharmaceutical composition required. For example, the physician orveterinarian could start doses of the compounds of the inventionemployed in the pharmaceutical composition at levels lower than thatrequired in order to achieve the desired therapeutic effect andgradually increase the dosage until the desired effect is achieved.

In particular embodiments, it is especially advantageous to formulatethe compound in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subjects tobe treated; each unit containing a predetermined quantity of therapeuticcompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical vehicle. The dosage unitforms of the invention are dictated by and directly dependent on (a) theunique characteristics of the therapeutic compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding/formulating such a therapeutic compound for thetreatment of a disease in a subject.

In one embodiment, the compositions of the invention are administered tothe subject in dosages that range from one to five times per day ormore. In another embodiment, the compositions of the invention areadministered to the subject in range of dosages that include, but arenot limited to, once every day, every two, days, every three days toonce a week, and once every two weeks. It will be readily apparent toone skilled in the art that the frequency of administration of thevarious combination compositions of the invention will vary from subjectto subject depending on many factors including, but not limited to, age,disease or disorder to be treated, gender, overall health, and otherfactors. Thus, the invention should not be construed to be limited toany particular dosage regime and the precise dosage and composition tobe administered to any subject will be determined by the attendingphysical taking all other factors about the subject into account.

Compounds of the invention for administration may be in the range offrom about 1 mg to about 10,000 mg, about 20 mg to about 9,500 mg, about40 mg to about 9,000 mg, about 75 mg to about 8,500 mg, about 150 mg toabout 7,500 mg, about 200 mg to about 7,000 mg, about 3050 mg to about6,000 mg, about 500 mg to about 5,000 mg, about 750 mg to about 4,000mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 50 mg toabout 1,000 mg, about 75 mg to about 900 mg, about 100 mg to about 800mg, about 250 mg to about 750 mg, about 300 mg to about 600 mg, about400 mg to about 500 mg, and any and all whole or partial incrementsthere between.

In some embodiments, the dose of a compound of the invention is fromabout 1 mg and about 2,500 mg. In some embodiments, a dose of a compoundof the invention used in compositions described herein is less thanabout 10,000 mg, or less than about 8,000 mg, or less than about 6,000mg, or less than about 5,000 mg, or less than about 3,000 mg, or lessthan about 2,000 mg, or less than about 1,000 mg, or less than about 500mg, or less than about 200 mg, or less than about 50 mg. Similarly, insome embodiments, a dose of a second compound (i.e., a drug used fortreating the same or another disease as that treated by the compositionsof the invention) as described herein is less than about 1,000 mg, orless than about 800 mg, or less than about 600 mg, or less than about500 mg, or less than about 400 mg, or less than about 300 mg, or lessthan about 200 mg, or less than about 100 mg, or less than about 50 mg,or less than about 40 mg, or less than about 30 mg, or less than about25 mg, or less than about 20 mg, or less than about 15 mg, or less thanabout 10 mg, or less than about 5 mg, or less than about 2 mg, or lessthan about 1 mg, or less than about 0.5 mg, and any and all whole orpartial increments thereof.

In one embodiment, the present invention is directed to a packagedpharmaceutical composition comprising a container holding atherapeutically effective amount of a compound or conjugate of theinvention, alone or in combination with a second pharmaceutical agent;and instructions for using the compound or conjugate to treat, prevent,or reduce one or more symptoms of a disease in a subject.

The term “container” includes any receptacle for holding thepharmaceutical composition. For example, in one embodiment, thecontainer is the packaging that contains the pharmaceutical composition.In other embodiments, the container is not the packaging that containsthe pharmaceutical composition, i.e., the container is a receptacle,such as a box or vial that contains the packaged pharmaceuticalcomposition or unpackaged pharmaceutical composition and theinstructions for use of the pharmaceutical composition. Moreover,packaging techniques are well known in the art. It should be understoodthat the instructions for use of the pharmaceutical composition may becontained on the packaging containing the pharmaceutical composition,and as such the instructions form an increased functional relationshipto the packaged product. However, it should be understood that theinstructions may contain information pertaining to the compound'sability to perform its intended function, e.g., treating or preventing adisease in a subject, or delivering an imaging or diagnostic agent to asubject.

Controlled Release Formulations and Drug Delivery Systems

Controlled- or sustained-release formulations of a pharmaceuticalcomposition of the invention may be made using conventional technology,using for example proteins equipped with pH sensitive domains orprotease-cleavable fragments. In some cases, the dosage forms to be usedcan be provided as slow or controlled-release of one or more activeingredients therein using, for example, hydropropylmethyl cellulose,other polymer matrices, gels, permeable membranes, osmotic systems,multilayer coatings, micro-particles, liposomes, or microspheres or acombination thereof to provide the desired release profile in varyingproportions. Suitable controlled-release formulations known to those ofordinary skill in the art, including those described herein, can bereadily selected for use with the pharmaceutical compositions of theinvention. Thus, single unit dosage forms suitable for oraladministration, such as tablets, capsules, gel-caps, and caplets, whichare adapted for controlled-release are encompassed by the presentinvention.

Most controlled-release pharmaceutical products have a common goal ofimproving drug therapy over that achieved by their non-controlledcounterparts. Ideally, the use of an optimally designedcontrolled-release preparation in medical treatment is characterized bya minimum of drug substance being employed to cure or control thecondition in a minimum amount of time. Advantages of controlled-releaseformulations include extended activity of the drug, reduced dosagefrequency, and increased subject compliance. In addition,controlled-release formulations can be used to affect the time of onsetof action or other characteristics, such as blood level of the drug, andthus can affect the occurrence of side effects.

Most controlled-release formulations are designed to initially releasean amount of drug that promptly produces the desired therapeutic effect,and gradually and continually release of other amounts of drug tomaintain this level of therapeutic effect over an extended period oftime. In order to maintain this constant level of drug in the body, thedrug must be released from the dosage form at a rate that will replacethe amount of drug being metabolized and excreted from the body.

Controlled-release of an active ingredient can be stimulated by variousinducers, for example pH, temperature, enzymes, water or otherphysiological conditions or compounds. The term “controlled-releasecomponent” in the context of the present invention is defined herein asa compound or compounds, including, but not limited to, polymers,polymer matrices, gels, permeable membranes, liposomes, or microspheresor a combination thereof that facilitates the controlled-release of theactive ingredient.

In certain embodiments, the formulations of the present invention maybe, but are not limited to, short-term, rapid-offset, as well ascontrolled, for example, sustained release, delayed release andpulsatile release formulations.

The term sustained release is used in its conventional sense to refer toa drug formulation that provides for gradual release of a drug over anextended period of time, and that may, although not necessarily, resultin substantially constant blood levels of a drug over an extended timeperiod. The period of time may be as long as a month or more and shouldbe a release that is longer that the same amount of agent administeredin bolus form.

For sustained release, the compounds may be formulated with a suitablepolymer or hydrophobic material that provides sustained releaseproperties to the compounds. As such, the compounds for use the methodof the invention may be administered in the form of microparticles, forexample, by injection or in the form of wafers or discs by implantation.

In a preferred embodiment of the invention, the compounds of theinvention are administered to a subject, alone or in combination withanother pharmaceutical agent, using a sustained release formulation.

The term delayed release is used herein in its conventional sense torefer to a drug formulation that provides for an initial release of thedrug after some delay following drug administration and that mat,although not necessarily, includes a delay of from about 10 minutes upto about 12 hours.

The term pulsatile release is used herein in its conventional sense torefer to a drug formulation that provides release of the drug in such away as to produce pulsed plasma profiles of the drug after drugadministration.

The term immediate release is used in its conventional sense to refer toa drug formulation that provides for release of the drug immediatelyafter drug administration.

As used herein, short-term refers to any period of time up to andincluding about 8 hours, about 7 hours, about 6 hours, about 5 hours,about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40minutes, about 20 minutes, or about 10 minutes and any or all whole orpartial increments thereof after drug administration after drugadministration.

As used herein, rapid-offset refers to any period of time up to andincluding about 8 hours, about 7 hours, about 6 hours, about 5 hours,about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40minutes, about 20 minutes, or about 10 minutes, and any and all whole orpartial increments thereof after drug administration.

It is to be understood that wherever values and ranges are providedherein, all values and ranges encompassed by these values and ranges,are meant to be encompassed within the scope of the present invention.Moreover, all values that fall within these ranges, as well as the upperor lower limits of a range of values, are also contemplated by thepresent application.

EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexamples therefore, specifically point out the preferred embodiments ofthe present invention, and are not to be construed as limiting in anyway the remainder of the disclosure.

Example 1 Peptide Metabolism, Bioavailability, and Bioactivity of theSmall Peptides Derived from P78

Experiments were designed to identify metabolic sites within the P78fragment. Experiments were designed to determine whether shorter analogshave better bioavailability and maintain the same bioactivity comparedto P78 or PEDF. Experiments were also performed to assess the effect ofsite-specific mutations on peptide metabolism, bioavailability, andbioactivity of the small peptides derived from P78.

Briefly, analogs of P78 were designed and synthesized (Table 1). As anon-limiting example, the analogs can be synthesized by GenScript(GenScript USA Inc., Piscataway, N.J. 08854). Some of the analogsincluded peptide modifications including but not limited to altering thecharge of the peptides, altering peptide stability, generating shorterfragments (e.g., 29 mer, 17 mer molecules), changing two residues in a17-mer fragment to Isoleucine, changing two residues in a 17-merfragment to Alanine, changing four residues in a 17-mer fragment (2 Ile;2 Ala), addition of fatty acid modifications at the N-terminus andPegylation at the C-terminus of a 29 mer and a 17-mer fragment).

TABLE 1 Peptide analogs of P78 Length Modified Theoretical PeptideSEQUENCE #AA residues MW P-78 VLLSPLSVATALSALSLGADERIESIIHRALYYD 44 N4667.30 LISSPDIHGT (SEQ ID NO: 42) SpxA1VLLSPLSVATALSALSLGADERIESIIHR (SEQ ID 29 N 3020.48 NO: 44) 81-1SALSLGADERTESIIHR (SEQ ID NO: 1) 17 N 1855.04 81-2SAISIGADERIESIIHR (SEQ ID NO: 2) 17 L → I 1855.04 81-3SAISIGADARTASIIHR (SEQ ID NO: 3) 17 (L → 1)₂; 1738.97 (E → A)₂ 81-4{BA*}VLLSPLSVATALSAISIGADARTASIIHR 29 (L → I)₂; 3119.50 {PEG*}(SEQ ID NO: 4) (E → A)₂* 81-5 {BA*}SAISIGADARTASIIHR{PEG*} (SEQ ID 17(L → I)2; 1954.24 NO: 5) (E → A)2^(#) 81-6ALYYDLISSPDIHGT (COOH) (SEQ ID NO: 6) 15 N 1664.82 81-7VLLSPLSVATAL (NH2) (SEQ ID NO: 7) 12 N 1183.44 81-8SALSLGADERTES (SEQ ID NO: 8) 13 N 1335.38 81-9SAISIGADARTAS (SEQ ID NO: 9) 13 (L → I)₂; 1219.31 (E → A)₂ 81-10VLLSPLSVATALSAISIGADARTASIIHR (SEQ ID 29 (L → I)₂; 2904.38 NO: 10) (E →A)₂ 81-11 {BA*}SAISIGADERTESIIHRI{PEG*} (SEQ ID 17 (L → I)₂* NO: 11)81-12 {BA*}NFGYDLYRVRSSMSPTTNSALSLGADER 35 P60 + 81-1; TESIIHR{PEG*}BA (NH2); (SEQ ID NO: 12)  PEG-COOH 81-13SALSLGAAERTESIIHR (SEQ ID NO: 13) 17 D → A 1811.01 81-14SALSLGANERTESIIHR (SEQ ID NO: 14) 17 D → N 1854.04 81-15SALSLGADEAIESIIHR (SEQ ID NO: 15) 17 R → A 1769.92 81-16SADERTESIIHR (SEQ ID NO: 16) 12 G → S 1413.50 81-17SALSLFADERTESIIHR (SEQ ID NO: 17) 17 G → F 1945.15 81-18SALSL(L-1-NAL)ADERTESIIHR (SEQ ID NO: 18) 17 G → 1995.21 L-1-NAL 81-19FGADERTESIIHR (SEQ ID NO: 19) 13 L → F 1530.65 81-20L-1-NALGADERIESIIHR (SEQ ID NO: 20) 13 L → L-1-NAL *81-4:CH3-CH2-CH2-(CO-NH)-VLLSPLSVATALSAISIGADARTASIIHR-(CO-NH)-PEG2-CH2COOH^(#)8l-5: CH3-CH2-CH2-(CO-NH)-SAISIGADARTASIIHR-(CO-NH)-PEG2-CH2COOH;{BA*}: Butyric acid on N-terminus: {PEG*} = Fmoc-NH-PEG2-CH2COOH

The analogs were tested for their efficacy in reducing levels of TNFα,IFNγ, VEGF-A and increasing cell viability in in vitro assays. Efficacywas compared to both non-treated oxidative stressed controls and thecontrol standard P78.

In addition, bioavailability studies of the peptides in rodent eyes wereconducted and the results were compared to the control standard P78peptide. For example, the peptides were tested in bioavailabilitystudies in rodents at 1, 2, 4, 8 hrs (n=4 each peptide/time pt). Theanimals were given a single eye drop containing 2 μg of each peptide. Ateach time point the animals were sacrificed, vitreous harvested, andbioavailability assessed by Maldi TOF.

The results indicate that peptide 81-5 has the highest level of activityin all three in vitro tests—inflammation, angiogenesis, and cellviability with efficacy ranging between 15-41% greater than P78 and18-65% improvement to the control stressed samples.

Two of the truncated (17mer) peptides demonstrated biological activitythat is better than that observed with P78. Peptide 81-2 shows goodefficacy and bioavailability profiles of all the peptides tested. It is˜8-18% better than P78 and the control in the inflammatory andangiogenesis assays and 8-26% better than P78 and control in theviability assay. 81-5 is a strong candidate from the in vitro studies.The results of these experiments are summarized in Table 2.

TABLE 2 Summary of biological activity SEQ *THFα *TNFα *IFNγ *IFNγ *VEGF*VEGF *ATP *ATP Analogs residue changes % to C % to P78 % to C % to P78% to C % to P78 % to C % to P78 {circumflex over ( )}after 4 hr P-78 44N +2 0% −5% 0% −5% 0% +17% 0% 1200 ng/ml SpxA1 29 N −3% −5% −9% −5% −5%0% +5% −11%  920 ng/ml 81-1 17 N −3% −5% −4% +1% −8% −3% +9% −11% 1800ng/ml 81-2 17 M(2) −13% −15% −12% −8% −18% −14% +26% +8% 2000 ng/ml 81-317 M(4) −7% −8% −9% −4% −12% −7% +11% −6% 2280 ng/ml 81-4 29 M(4) −7%−8% −15% −11% −11% −6% +7% −9% 81-5 17 M(4) −18% −19% −20% −15% −20%−16% +65% +41% 81-6 15 N 81-7 12 N 81-8 13 N 81-9 13 M 81-10 29 M

Example 2 The Effects of the Peptides to Increase Cell Viability

The analogs disclosed in Table 1 were assayed for improved stability andefficacy in in vitro assays for inflammation, angiogenesis, and celldeath, which are the three leading pathological features of diabeticretinopathy.

Briefly, to test efficacy of the compounds in preventing cell death,experiments were performed to screen for the ability of each analogto: 1) increase cellular bioenergetic levels in an ATP assay and 2)reduce cell death in an LDH assay. To test the action of each analog oninflammatory processes, Luminex bead arrays were used to examine theability of each analog to decrease production of inflammatory cytokines.To test for the potential of each analog to reduce vascular leakage,qPCR was used to examine the ability of each analog to increase mRNAlevels of ZO1 and occludin, two junction proteins essential to vascularintegrity. To test peptide stability in the vitreous, the analogs wereincubated with dissected vitreous humor and the samples were analyzed atvarious time points using Maldi-TOF. These tests allowed for thesystematic selection of analogs having the least toxicity and thestrongest potential to reduce pathology in vivo.

Twenty P78 peptide analogs were generated. The size of the analogsranged from 35 to 12 amino acids in length (Table 1). These peptides hadinternal amino acid substitutions of the sequence and/or helixstabilization and hydrophobic cap changes to improve peptide stabilityand efficacy. The peptides were all tested and compared to P78 and spxA1for efficacy in cell viability and release of TNFα, IFNγ, andVEGFA—three of the most potent inflammatory, vascular leakage, andangiogenesis markers in the retina. Several of the peptides showedactivity differences ranging between ˜5-20% when compared to P78.Several of the peptides were significantly less active than P78.

The effects of the peptides to increase cell viability by increasinglevels of ATP were tested in two models of cell death: (i) serumstarvation; (ii) oxidative stress caused by hydrogen peroxide toxicity(300 uM). With respect to serum starvation, human ARPE19 cells weregrown to ˜85% confluency in 10% serum containing medium after which thecells were placed in serum-free medium for 48 hr in the presence orabsence (control) of 25 nM of each peptide. For ATP assay,CellTiter-Glo® reagents were used and luminescence was determined after12 min spectrophotometrically (n=4) (FIG. 2A). With respect to oxidativestress, human ARPE19 cells were grown to ˜85% confluency in 10% serumcontaining medium. The cells were then treated with 300 uM H₂O₂ for 48hours in the presence or absence (control) of 25 nM of each peptide.Levels of ATP were measured and % luminescence to controls and P78 wasquantitated (n=4). Changes to ATP levels are plotted against controlvalues and p values are given to P78 (FIG. 2B). The results demonstratethat peptides 81-2, 81-5, 81-12, 81-13, 81-20 are considered goodcandidates for the in vivo studies.

Example 3 The Effects of the Peptides on the Inflammatory and VascularPermeability/Angiogenesis Cytokines

Experiments were designed to assess the effects of each peptide comparedto P78 on the inflammatory and vascular permeability/angiogenesiscytokines TNFα, IFNγ, and VEGFA. Briefly, human retinal RPE cells weregrown to ˜75% confluency in 10% serum containing medium. The cells werethen exposed to serum starvation in the presence or absence (control) of25 nM of each peptide including the prototype P78 and the activefragment of P78, SpxA1. The supernatant from each treatment was testedfor effects on the secretion of TNFα, IFNγ, and VEGFA. The datarepresent the percent change to the control samples and p values aregiven for each peptide in relationship to the P78 treatment (FIGS. 3Athrough 3B).

The results presented herein also demonstrate that peptides 81-10 and81-19 have significantly better effects in reducing VEGFA levels butwere less effective than P78 on the other assays. These peptides may beconsidered specific anti-VEGF drugs.

Example 4 Ability to Reduce Vascular Leakage

To test for the potential of each peptide (81-2, 81-5, 81-12, 81-13 and81-20) to reduce vascular leakage, qPCR was used to examine the abilityof each analog to increase mRNA levels of ZO1, a junction proteinessential to vascular integrity (FIG. 4A). The results demonstrate thatthe peptides resulted in a higher expression of ZO1 compared to P78 andSpxA1.

Example 5 Peptide Stability

To test peptide stability in the vitreous, the analogs were incubatedwith dissected vitreous humor and the samples were analyzed at varioustime points using Maldi TOF (FIG. 4B). The results demonstrate that thepeptides were more stable compared to P78 and SpxA1.

Example 6 Structure of Peptides

CD spectroscopy was used to evaluate the structure of the peptides (FIG.5A-FIG. 5D). The CD spectra for the peptides demonstrate the helicalstructure of the peptides. It was observed that while all of thepeptides have some helical structure, 81-5 is almost a perfect for alphahelix.

Example 7 In Vivo Testing The peptides of the invention can be tested invivo in any animal model.

An exemplary animal model is the Ins2 ^(Akita) mouse described by Liu etal. (2012, Mol Med 18: 1387-1401), which is hereby incorporated byreference in its entirety. This animal model has been used to assess theactivity of PEDF in reducing diabetic retinopathy complications.

Eye Drop Formulation

Briefly, an eye drop formula containing 1 mg/mL labeled dialyzed peptidediluted in artificial tears (Prestige Brands International) is preparedfor each sample. Eye drops are administered in 5 μL (5 μg) volume ontothe ocular surface of rats or diabetic mice to test distribution of thelabeled peptides in the eye and to quantify levels reaching the vitreouscompartment.

Eye Drop Treatments: Ins2^(Akita) Diabetic Mice

Hyperglycemic heterozygote C57BL/6J Ins2Akita mice (The JacksonLaboratory, Bar Harbor, Me., USA) have a mutation in the Ins2 gene thatresults in hyperglycemia at ˜4.5 wks of age and detectable vascularcomplications at ˜16-17 wks of age (12-13 wks of hyperglycemia). Theseanimals are kept on a 12-h light-dark cycle, and food and water areprovided ad libitum. Insulin is not supplemented to the diet.Heterozygote Ins2Akita males are crossed with C57/B16/J females anddiabetic offspring are confirmed by genotyping and blood glucoselevels >250 mg/dL at 4.5 wks of age. Only male hyperglycemic mice areused, since diabetic retinopathy in females displays inconsistentpathological features. Glucose levels are measured in blood samplesobtained from tail punctures by using the One-Touch LifeScan meter(LifeScan, Milpitas, Calif., USA). Animals with 300-400 mg/dL serumglucose levels are used in this study.

One group of diabetic Ins2Akita mice are treated with eye dropscontaining labeled peptides for 2-4 h to examine distribution of themolecules in the eye. The animals are anesthetized at specific timepoints by using ketamine (100 mg/kg) and xylazine (10 mg/kg)intraperitoneally, and eyes are enucleated and washed extensively inice-cold PBS. Whole eyes or dissected retinas and corneas are fixed with4% paraformaldehyde, embedded in O.C.T. (Tissue-Tek; Sakura, Alphen aanden Rijn, the Netherlands) and are cryosectioned.

A second group of mice is used to determine effects of the peptides ondiabetes-induced pathologies in the retina over a period of 15 wks ofhyperglycemia. This time point was chosen because vascular pathology iseasier to detect by the present methods (beginning at ˜12.5 wks).Diabetic male Ins2Akita mice will receive one peptide eye drop per weekfor 13-15 wks immediately at the onset of hyperglycemia (4.5 wks old).Mice are manually restrained or lightly anesthetized when necessary, and5-μL unlabeled peptides in ATs are applied to the corneal surface. Thisgroup is subdivided into four treatment groups, each receiving one ofthe following eye drops: P60, P78, P78+P60 or ATs. Both eyes willreceive the same treatment to avoid drug cross- contamination betweenthe eyes by the animal. Animals are given 200 μL salineintraperitoneally to prevent dehydration and are placed in warmed cagesto recover.

Presented herein are experiments to evaluate the effects of the peptidesof the invention on cell death, vascular leakage, and inflammation.These assays may be used to evaluate the peptides in treating diabeticretinopathy, glaucoma, retinitis pigmentosa, AMD, and ROP.

Inflammation

The effects of the peptides on inflammation is evaluated by measuringthe reduction of levels of inflammatory cytokines TNFα and Interferongamma in the vitreous using the Affymetric Biolplex Luminex bead assayand reduction of microglia activation as determined by IBA 1immunolabeling of microglia in the retina

Cell Death

The effects of the peptides on cell death is evaluated by estimatingnumber of cells undergoing apoptosis by TUNEL assay and cell dropoutusing staining by propidium iodide or DAPI. For evaluation of thepeptides in the treatment of diabetic retinopathy, glaucoma, or ROP,cell death is measured in retinal ganglion cells. For evaluation ofRetinitis pigmentosa, cell death is measured for photoreceptors. Forevaluation of AMD, cell death is measured for photoreceptors and RPEcells.

Vascular Leakage (Macular Edema)

Leakage is measured by live imaging of the retina. Mice are anesthetizedusing Ketamine 100 mg/Kg, Xylazine 10 mg/Kg, ocular surface lubricatedwith Optive Lubricant (Allergan), pupils dilated with 0.5% TropicamideOphthalmic Solution (Alcon), and animals injected with 100 ul AK-FLUORsolution (10%). Fluorescein images of the retina are acquired using thePhoenix Micron III retinal imager and extent of vascular leakage in theretina is estimated by counts of fluorescent areas. Fluorescentintensity is measured using the NIH Image J software.

Paraffin sections of the retina is also immunolabeled with antibodiesfor two tight junction proteins in the vasculature—ZO1 and Occludin,which are important to maintaining vessel integrity.

Vitreous levels of VEGF in the diseased retina are measured using theAffymetrix Luminex bead assay and compared to untreated controls andwild type animals. VEGF levels are also be measured by western blotanalysis to confirm the luminex bead assays

Vascular leakage is also evaluated by estimating pericyte drop out.

To evaluate the peptides for treatment of diabetic retinopathy or ROP,vascular leakage is examined in the inner retina. To evaluate thepeptides for treatment of AMD, vascular leakage is examined in thechoroidal vasculature and outer retina.

Example 8 Bioavailability and Efficacy of Peptide Analogs in In VivoModels

From the in vitro P78 peptide analog screening studies, fivestructurally diverse analogs with best biological activity were selectedto test their efficacy in the diabetic mouse eye. In vivo effects ofthese peptides were tested in the Ins2Akita mouse model of DR, awell-characterized DR model that has a mutation in the insulin gene.Heterozygote Ins2Akita mice (Jackson Lab) become hyperglycemic (HG) at˜4.5 wks of age. RGC death and inflammation occur within the first 4-6wks of DR and vascular pathology is first noticed ˜13 wks after theonset of diabetes. The studies were designed to identify peptidecandidates with pleotrophic effects on cell death, inflammation, andvascular leakage in the diabetic retina.

Male hyperglycemic mice with blood glucose levels >300 mg/dL were usedand treatments carried as previously described for P78 (Liu et al.,2012, Mol Med., 18(1): 1387-1401). Diabetic mice were treated 2×/wk for15 wks at the onset of HG using a single dose of 5 μg/5 μl artificialtears for each drug, a dose at which P78 is effective. Both eyesreceived the same treatment to avoid drug cross contamination betweenthe eyes of an individual animal. At the end of the longitudinalstudies, eyes were enucleated from anesthesized animals, vitreouscollected, and retina dissected and embedded in paraffin formorphometric analysis of RGC survival and vascular leakage.

Before the longitudinal efficacy studies were carried out in vivo, thebioavailability of the five analogs was examined in comparison to theparent compound P78 and its active 29-mer derivative, SpxA1.

Peptide Bioavailability in the Retina

Bioavailability of each peptide was determined in vitreous samplesobtained at various time points after eye drops were given. Vitreoussamples were harvested from enucleated eyes and immediately analyzed bymass spectrometry. Spectral intensities were compared to spectra ofknown concentrations of each analog to calculate amounts of peptidesreaching the vitreous. The eye drops administered to normal mouse eyescontained 5 μg of the test compound. Bioavailability was studied atthree time points: 1, 4, 6 hr using four animals/time point (n=8 eyes).The vitreous was rapidly dissected from each group after the drops weregiven and immediately analyzed by MALDI TOF. Several of the analogsshowed significantly better access to the retina compared to P78 andSpxA1 but all showed peak levels at approximately 1 hr after the dropswere administered. Analog 81-5 showed the best stability profile in thevitreous after 6 hr compared to the parent compound and its truncated29-mer SpxA1. The quantitative bioavailability data obtained by MassSpectrometry are shown in FIG. 6.

The amount of peptides reaching the retina 1 hr after eye dropadministration was also visualized by confocal microscopy. In thisexperiment, eye drops containing the peptides were given to the diabeticIns2Akita mice for 1 hr. The animals were then euthanized, eyesdissected, fixed in 4% paraformaldehyde overnight, and whole globes weresectioned in OCT. Sections were then immunolabeled with an antibodyagain the P78 peptide, which also recognizes the full length PEDF andthe analogs. As controls, C57BL/6 (wt) and diabetic Ins2Akita micetreated with vehicle (Diabetic Control) were immunolabeled to comparewith the peptide treated groups. The data in FIG. 7 shows weakendogenous PEDF staining in the wt retina and an upregulation ofendogeneous PEDF in the diabetic control retinas suggesting that indiabetes, PEDF is upregulated, possibly as an endogeneous therapeuticapproach by the eye. The intense labeling after the peptide eye dropswere given largely represents the peptide analogs present in the retinaand indicates that a significant amount of the peptides is delivered tothe retina by topical routes. Staining is visible throughout the retina,but is more strongly seen in the choroid and RPE-Photoreceptor layers ofthe retina. The intense labeling seen in the choroidal vasculaturesuggests that these vessels may be a route of delivery of these peptides(FIG. 7). Thus, both the mass spectrometry and immunolabeling studiesprovide strong evidence that these small therapeutic peptides can bedelivered to the back of the eye when administered topically and isrepresents a non-inOvasive approach to treating retinal diseases.

Reduction of Inflammation

Vitreous samples harvested at the termination of the 15 weeks efficacystudy were analyzed to detect levels of proinflammatory cytokines usingthe Bioplex multiplex platform. This system utilizes polystyrene luminexbead arrays and the xMAP technology. Target inflammatory markersexamined were TNFα, IFNγ, and IL-6, and the major proangiogenesiscytokine, VEGFA. FIG. 8 depicts the expected rise of the proinflammatorycytokines in the diabetic retina compared to wt samples and a markedreduction in all three by the P78 peptide analogs, mimicking the resultsof the in vitro studies. While the peptides showed similar effects inreducing TNFα and IFNγ, 81-5 and 81-13 showed slightly but significantlybetter effects in reducing IL-6 (FIG. 8).

VEGF Levels

Levels of VEGF were also quantitated in the vitreous of the 15 wkspeptide treated and control groups using the Luminex bead technology.From this experiment, a significant increase in VEGF levels wasobserved, as shown before (Liu et al., 2012, Mol Med, 18(1): 1387-1401)in the vehicle treated diabetic controls compared to the age and weightmatched wild type C57BL/6. All peptide treatments resulted in reducedlevels of VEGF in the vitreous relative to the diabetic controls withanalogs 81-5 and 81-12 slightly but significantly more effective (FIG.9).

Reduction in Vascular Leakage

Vascular hemorrhaging was quantitated by measuring extent of albuminextravasation in the retina by leaky retinal vessels. An ELISA approach(FIG. 10) was used to quantitate extent of albumin leakage from bloodvessels into the retina parenchyma, and confocal microscopy wasperformed using an antibody to mouse albumin to detect changes inalbumin levels in the controls and treatment groups (FIG. 11). FIG. 10demonstrates that there was a significant increase in vascular leakagein the diabetic control retinas compared to the wild type animals at 15weeks of diabetes. The selected panel of analogs was also effective inreducing albumin content in the retina with Spx, 81-2 and 81-5 having asmall but significant advantage over the other analogs. This analysiswas confirmed by microscopic evidence on retinal sections immunolabeledfor albumin. From this study, it was evident that the diabetic retinascontained higher levels of albumin throughout the retinal parenchyma andin blood vessels (arrows) as show in FIG. 11 (20×). Higher magnification(FIG. 12) of these retinal images (40×) shows increased albumin levelsin the photoreceptor inner and outer segment areas, vascular leakageinto the retinal parenchyma in the outer plexiform layers (OPL; FIG.12A, arrow) and the retinal ganglion cell layer (RGC; FIG. 12B-FIG. 12C,FIG. 12E-FIG. 12F; arrows), and a large blood vessel in the RGC layer(FIG. 12D). The data suggest that not only is there leakage from themicrovessels in the inner retina but that the RPE-Choroidal vasculatureadjacent to the photoreceptor layer maybe compromised in diabeticretinopathy. In FIG. 13, the effects of the peptides on vascular leakageas measured by albumin extravasation into the retina are shown. Allpeptide-treated retinas immunolabeled with the albumin antibody showedless fluorescence intensity throughout the retina including thephotoreceptor IS/OS compared to the diabetic controls. Albumin levelswere comparable to the wild type retinas although inner retinalmicrovessels appeared larger than the wt. The microscopy data thusconfirms the ELISA quantitative measurements (FIG. 10) which argued forincreased vascular leakage in the diabetic retina and a reduction afterpeptide treatments.

RGC Survival

RGC loss occurs early in diabetic retinopathy in both humans androdents. In this study retinal sections from peptide treated and controlgroups were stained with DAPI to manually count the nuclei of survivingRGCs. Six non-serial sections from each eye in the peripheral andcentral retinas were used for morphometric analysis. Stained nuclei inthe RGC layer were counted in all retinal eccentricities using high-resolution confocal optical slices of DAPI stained retinas. Cell countswere taken from 6×250 mm zones along the length of the retina fromcentrally located fields adjacent to the optic nerve to the periphery. 6fields/retina were analyzed and data presented as the avg # cells/400μM. (n=6). The results of this experiment are presented in FIG. 14-FIG.15. The morphometrics data in FIG. 14 show a decrease in the number ofsurviving retinal ganglion cells (RGCs) in untreated diabetic controlretinas (DC) as previously demonstrated. The extent of theneuroprotective actions of the peptide treatments on these cells wasalso significant for each treatment and comparable RGC counts in thewild type retina. The efficacy profiles of this group of peptides forRGC survival are comparable and suggest that this set of P78 analogs areclearly potent in preventing diabetes-induced degeneration of RGC cells.Whether the neuroprotective effect of the analogs is a direct action onRGCs themselves or is indirectly mediated through the reduction ofproinflammatory cytokines is worthy of further investigation. Althoughthe peptide analogs are structurally diverse, the modifications wereconservative. Although they are much shorter fragments that P78, eachcontain the same core group of peptides. These peptides were selectedbecause of their in vitro activity was superior to P78. Without a doubt,the in vitro and in vivo efficacy profiles indicate that these peptidescontain the active core sequence of P78 and constitute a panel oftherapeutic analogs for DR.

The micrographs in FIG. 15, are representative images of DAPI and TUNELstained retinas of peptide treated diabetic mice and control groups. TheRGC layer is indicated as the single cell layer in the inner retina.DAPI staining was used to count the nuclei in the RGC layer and TUNELassay (green fluorescence) was used to detect ongoing cell death aftertreatment. Several of the peptides showed little ongoing cell death inthe RGC layer and increased numbers of surviving RGCs compared to thevehicle treated diabetic animals. Of interest is the abnormal morphologyand disorganization of the nuclei comprising the inner nuclear layer(INL) in the untreated diabetic group. The INL also appears to beaffected by the peptide treatments and is shown to be more like the wtafter treatment.

Bioavailability of Peptides in Primate Eyes

From the studies using the DR mouse model, two active, structurallydiverse peptides 81-5 and 81-13 were selected to test their availabilityin the primate eye when given topically. There are substantialdifferences between the eyes of rodents, primates, and humans. The mostobvious is size of the aqueous and vitreous fluid compartments. Fluidflow forward from the ciliary body in primates/humans is countercurrentto drug movement to the back of the eye and may influence drugconcentrations in the vitreous. In this study, it was examined whetherthe selected compounds had access to the retina when given topically toprimate eyes as they do in rodents. Peptide doses used were scaled up by8-20 fold as an estimate to account for differences in eye volumebetween rodents and primates.

Adult males and females were equally distributed into 3 treatment groups(Table 3) and randomized by weight criteria. Animals were fastedovernight then sedated with ketamine (8 mg/kg, I.M) and xylazine (1.6mg/kg, I.M) prior to all procedures. Two monkeys received a topical doseof 40 μg (40 μl ) of 81-5 OU (both eyes) and underwent vitreocentesis at˜1 hr (Group 1) after dosing. Four monkeys received a topical dose of100 μg (50 μl) of 81-13 OU and underwent vitreocentesis at ˜1 hr (Group2; 2 animals) or ˜2 hr (group 3; 2 animals) after dosing (Table 3).Animals remained under continuous sedation prior to vitreous humorcollection. Eyes were manually blinked (4 blinks/min) for 2 minutesafter dosing to mimic ocular delivery in a non-sedated animal. Prior tovitreous humor collection, topical local anesthesia was administered(0.5% proparacaine) and eyes disinfected with 5% Betadine. A 25-gauge,0.5 inch needle was placed 2 mm posterior to the limbus in the inferiortemporal quadrant, targeting the central vitreous. A volume of 100 μL ofvitreous humor was aspirated gently from the right (OD) and left (OS)eyes and transferred to labeled pre-tared cryovials. A larger volume ofvitreous humor (200 μl) was withdrawn into the syringe from animal X429because the operator had to pull harder to overcome the viscosity ofvitreous. The cryovials were flash frozen in liquid nitrogen. Vitreoushumor collection was followed by topical administration of a tripleantibiotic ointment (neomycin/polymyxin B sulfates/bacitracin zinc).Animals were returned to the colony after vitreocentesis. Since botheyes were used for the same peptide, the n value was 4 for eachpeptide/dose/time.

TABLE 3 Peptide bioavailability in primate (Vervets) eyes Body Vitreoushumor Mass Spec Animal weight Test Dose/ collected after Volume Avg ConcGroup ID Sex (kg) Eye article Topical dosing collected ng/ml 1 Z998 Male6.54 OU 81-5  40 μg/40 μL/eye OD: 67 min OD: 100 μL OS: 69 min OS: 100μL 1 K099 Female 3.78 OU 81-5  40 μg/40 μL/eye OD: 65 min OD: 100 μLGroup 1(n = 4) OS: 66 min OS: 100 μL 158.5 ± 15.5 2 V715 Female 4.68 OU81-13 100 μg/50 μL/eye OD: 59 min OD: 100 μL OS: 60 min OS: 100 μL 2K146 Male 5.92 OU 81-13 100 μg/50 μL/eye OD: 58 min OD: 100 μL Group 2(n = 4) OS: 64 min OS: 100 μL 237.5 ± 32.5 3 X429 Female 4.98 OU 81-13100 μg/50 μL/eye OD: 120 min OD: 100 μL OS: 118 min OS: 200 μL 3 K169Male 8.28 OU 81-13 100 μg/50 μL/eye OD: 125 min OD: 100 μL Group 3 (n =4) OS: 128 ml OS: 100 μL 212.5 ± 15.0Samples were analyzed by mass spectrometry and concentrations calculatedusing a standard curve that plotted intensity vs known concentration.Although the levels of the peptides reaching the vitreous compartment inthe primate eye reached therapeutic levels (bioactivity=20-50 ng/ml),peptide concentrations were lowered by ˜2 fold than that seen in therodent eyes possibly because of the drug:volume ratio and thecountercurrent fluid flow forward in the anterior chamber of the eye.However, the study suggested that increasing dosage may result inincreased concentrations of the drugs in the vitreous compartment asobserved with peptide 81-13 which showed comparable bioavailabilityprofiles with 81-5 in rodents. The peptide concentration in the vitreouswas not significantly different between the 1 and 2 hr treatment and waseven a bit lower at 2 hr, suggesting that peak concentrations weresimilar to the rodent at 1 hr after topical administration.

In summary, the in vivo data in diabetic mice indicate that the selectedset of P78 analogs that showed the best activity in vitro were alsoactive in vivo in reducing hallmark pathologies of diabetic retinopathy,namely inflammation, vascular leakage, and cell degeneration. While theminor differences in efficacy in vivo was unexpected, these structurallydiverse molecules represent a therapeutic panel of active compounds fordiabetic retinopathy and have advantages for the development of moreeffective next generation compounds. The primate ocular bioavailabilitystudy is very encouraging and holds promise for delivery of these smalltherapeutic peptides to the human eye to treat ocular diseases.

Example 9 Regulation of Endothelial Tube Formation—An Angiogenesis Assay

Human dermal fibroblasts were cultured at 1×10⁵/well in IncuCyte seedingmedium (Essen Bioscience) in 96 wells plates for 1 hr. Medium wasremoved and replaced with complete IncuCyte growth medium (EssenBioscience). Human umbilical vein endothelial cells (HUVEC) containing agreen fluorescent marker (HUVEC CytoLight green) cells at ˜2000cells/well were /well to attached dermal fibroblasts cultures and placedin the IncuCyte Zoom fluorescent and phase contrast multiplex imagingincubator (Essen BioScience). Twenty four hours later 4 ng/ml VEGF wasadded to all wells and 100 nM/ml PEDF peptides added to test wells.Cultures containing VEGF alone were used as positive controls and thosecontaining 100 μM Suramin (VEGF inhibitor) used as negative controls.The cultures were placed at 37° C. in the Incucyte Zoom and HUVEC tubelength networks measured over a 12 day incubation period using theIncucyte CellPlayer Angiogenesis software module and interface systems.Media was refreshed every 3-4 days.

Formation of tubes by HUVEC was inhibited by the selected peptidescompared to VEGF. The most potent were P78, the parent PEDF activepeptide, 81-2, and 81-5. The other three peptides: 81-12, 81-13, and81-20 prevented tube formation compared to VEGF but less effective.Representative micrographs of HUVEC tube induction for VEGF alone and100 μM Suramin treatment are shown in FIG. 16A, while micrographs ofHUVEC tube formation induction for peptide treatments are shown in FIG.16B. Quantitative data of triplicate experiments is shown in FIG. 16C.

Example 10 Transcription Factor Targets of PEDF-Derived Peptides

Activation of transcription factors by P78, spx or 81-5 was measured inhuman RPE cells. The Affymetrix Procarta Multiplex Transcription factorpanel (Affymetrix, Inc. Santa Clara, Calif.) was used to obtainquantitative measurement of DNA binding activity of 44 transcriptionfactors. Cells were seeded at 1×10⁵ cells/ml in 6 well plates. Cellswere treated with 50 nM peptides in 2% FBS medium for 48 hr, peptideswere supplied twice after 24 hr. Cells were harvested using Buffer Aworking reagent (250 μl/well) (1ml Buffer A: 10 μl DTT, 10 μl proteaseinhibitor, 10 μl phosphatase inhibitor I, 10μ1 phosphatase inhibitor II)and centrifuged at 14,000×g for 3 min at 4° C. Supernatant containingcytoplasmic proteins was collected and protein concentration estimatedusing the BioRad DC protein assay. To the nuclear pellets, 30 μl/wellbuffer B (1 ml Buffer B: 10 μl DTT, 10 μl protease inhibitor, 10 μlphosphatase inhibitor I) was added and the samples incubated for 2 hoursat 200 rpm. Supernatant containing nuclear proteins was collected bycentrifugation at 14,000×g for 5 min at 4° C. and protein concentrationmeasured using Bio-Rad DC protein assay. To form transcription factor(TF) protein/DNA complexes, 2 μg (5 μl) nuclear or cytoplasmic proteins,10 μl biotin-labeled DNA binding probes (Cis elements; Detection probes,TF panel 2) and 5 μl of nuclease-free distilled water were added to eachwell of a PCR plate and incubated at 15° C. for 30 min using a PCRmachine. 20 μl ice-cold binding buffer was added to each well containingthe TF-DNA complexes and 30 μl sample from each well transferred to aprewashed separation plate and incubated on ice for 30 min. The TF-DNAcomplexes were bound to a semi-porous filter using a separation plate.Unbound biotin-labeled DNA binding probes and proteins were removed bywashing and the DNA complexes were collected by centrifugation using563×g for 3 min at 4° C. DNA complexes were denatured at 95° C. for 5min using a PCR machine, then added to prewashed TF-specific antisenseconjugated beads (capture beads, TF panel 2, Affymetrix), and incubatedfor 10 min at RT with constant shaking at 500 rpm. Samples were thentransferred to a 50° C. incubator for 30 min without shaking. Beads weresubsequently washed by vacuum filtration and incubated withstreptavidin-PE (Affymetrix) for 30 min at room temperature with shakingat 500 rpm. The beads were washed again, resuspended with readingbuffer, and incubated for 5 min at room temperature. DNA bindingactivity of the transcription factors was measured using the Bio-Plex200 system (bio-Rad) and the xMAP technology.

P78 regulated transcription factors were also identified in the mouseretina. Diabetic Ins2Akita mice were infused with 0.5 mg/kg/day P78 for6 weeks constant infusion using Alzet pumps. Controls were given PBSalone. Retinas of age-matched normal C57BL/6, diabetic mice treated withvehicle alone, and P78 treated animals were dissected and nuclearextracts collected.

It was observed that PEDF and the active peptide region (P78) or atruncated sequence of this region (spx) regulates transcription factors(TFs) that control expression of a wide range of genes that are involvedin varied and complementary processes (FIG. 17). These include TFsregulating genotoxic stress, environmental stress, and apoptosis (HiNF,Nrf1), cell cycle progression and cell differentiation (RB), and TFscontrolling ER stress and inflammatory response (CCAAT/C-EBP).

Example 11 Regulation of Inflammatory Cytokines by PEDF-Derived Peptides

Human Muller glia cells (MIO-M1) were seeded out at a density of 1×10⁵in DMEM growth medium. Cultures were treated with 25 nM peptides at thetime of seeding for 48 hours. Conditioned medium was harvested bycentrifugation and 50 μL was analyzed using a focused proteomicsapproach of a multiplex panel of 44 inflammatory Luminex cytokine(Affymetrix Procarta cytokine profiling panel). Analysis was carried outusing the xMAP technology and BioRad Manager 4.1 software.

The analysis (FIG. 18A and FIG. 18B) confirms the effects of theselected panel of active PEDF peptides on the regulation ofpro-inflammatory cytokines in human RPE cells and the diabetic retina.These results are consistent with previous findings in human RPE cellsand in vivo work, and suggest that Muller glia cells are a key target ofthe peptides.

In the retina, Muller Glia cells are highly reactive to retinal injuryincluding those caused by rises in glucose levels. Their reactivity isused as a molecular index of pathological occurrences in the retina. Themajor proinflammatory cytokines regulated include IFN-γ, TNF-60 , IL-2,IL-5 (FIG. 18A) with the peptides showing varied effects on thesecytokines but several were more effective than the parent P78 peptidefragment. In addition, these cells are a major producer of VEGF in theretina and these peptides show better effects than P78 in reducing itsproduction levels by these cells (FIG. 18A). This data again suggeststhat a major target of the PEDF peptide analogs is towards inflammatorymolecules and VEGF in the retina.

Example 12 Peptide and Nucleic Acid Sequences

Presented herein are the peptide sequences and the calculated nucleicacid sequences for the peptides. The amino acid sequences werecalculated using EMBOSS Backtranambig—a program that reads a proteinsequence and writes the nucleic acid sequence it could have come from.It does this by using nucleotide ambiguity codes that represent allpossible codons for each amino acid (Table 4).

SpxA1 (Spx) amino acid sequence:  (SEQ ID NO: 44)VLLSPLSVATALSALSLGADERTESIIHR nucleic acid sequence: (SEQ ID NO: 45)GTNYTNYTNWSNCCNYTNWSNGTNGCNACNGCNYTNWSNGCNYTNWSNYTNGGNGCNGAYGARIVIGNACNGARWSNATHATHCAYMGN 81-1 peptide- (SEQ ID NO: 1)SALSLGADERTESIIHR nucleic acid-  (SEQ ID NO: 21)WSNGCNYTNWSNYTNGGNGCNGAYGARIVIGNACNGARWSNATHATHCAY MGN 81-2 peptide-(SEQ ID NO: 2) SAISIGADERTESIIHR nucleic acid- (SEQ ID NO: 22)WSNGCNATHWSNATHGGNGCNGAYGARIVIGNACNGARWSNATHATHCAY MGNTH 81-3 peptide-(SEQ ID NO: 3) SAISIGADARTASIIHR nucleic acid- (SEQ ID NO: 23)WSNGCNATHWSNATHGGNGCNGAYGCNIVIGNACNGCNWSNATHATHCAY MGNN 81-4 peptide-(SEQ ID NO: 4) {BA*}VLLSPLSVATALSAISIGADARTASIIHR{PEG*} nucleic acid-(SEQ ID NO: 24) GTNYTNYTNWSNCCNYTNWSNGTNGCNACNGCNYTNWSNGCNATHWSNATHGGNGCNGAYGCNMGNACNGCNWSNATHATHCAYMGN 81-5 peptide - (SEQ ID NO: 5){BA*}SAISIGADARTASIIHR{PEG*} nucleic acid- (SEQ ID NO: 25)WSNGCNATHWSNATHGGNGCNGAYGCNIVIGNACNGCNWSNATHATHCAY MGN  81-6 peptide-(SEQ ID NO: 6) ALYYDLISSPDIHGT nucleic acid- (SEQ ID NO: 26)GCNYTNTAYTAYGAYYTNATHWSNWSNCCNGAYATHCAYGGNACN 81-7 peptide-(SEQ ID NO: 7) VLLSPLSVATAL nucleic acid- (SEQ ID NO: 27)GTNYTNYTNWSNCCNYTNWSNGTNGCNACNGCNYTN 81-8 peptide- (SEQ ID NO: 8)SALSLGADERTES nucleic acid- (SEQ ID NO: 28)WSNGCNYTNWSNYTNGGNGCNGAYGARMGNACNGARWSN 81-9 peptide- (SEQ ID NO: 9)SAISIGADARTAS nucleic acid- (SEQ ID NO: 29)WSNGCNATHWSNATHGGNGCNGAYGCNMGNACNGCNWSN 81-10 peptide- (SEQ ID NO: 10)VLLSPLSVATALSAISIGADARTASIIHR nucleic acid- (SEQ ID NO: 30)GTNYTNYTNWSNCCNYTNWSNGTNGCNACNGCNYTNWSNGCNATHWSNATHGGNGCNGAYGCNMGNACNGCNWSNATHATHCAYMGN 81-11 peptide-  (SEQ ID NO: 11){BA*}SAISIGADERTESIIHR{PEG*} nucleic acid- (SEQ ID NO: 31)WSNGCNATHWSNATHGGNGCNGAYGARNIGNACNGARWSNATHATHCAYM GN  81-12 peptide-(SEQ ID NO: 12) {BA*}NFGYDLYRVRSSMSPTTNSALSLGADERTESIIHR{PEG*}nucleic acid-  (SEQ ID NO: 32)AAYTTYGGNTAYGAYYTNTAYMGNGTNMGNWSNWSNATGWSNCCNACNACNAAYWSNGCNYTNWSNYTNGGNGCNGAYGARNIGNACNGARWSNATHATH CAYMGN 81-13 peptide-(SEQ ID NO: 13) SALSLGAAERTESIIHR nucleic acid- (SEQ ID NO: 33)WSNGCNYTNWSNYTNGGNGCNGCNGARMGNACNGARWSNATHATHCAYMG N  81-14 peptide-(SEQ ID NO: 14) SALSLGANERTESIIHR nucleic acid- (SEQ ID NO: 34)WSNGCNYTNWSNYTNGGNGCNAAYGARMGNACNGARWSNATHATHCAYMG N  81-15 peptide-(SEQ ID NO: 15) SALSLGADEATESIIHR nucleic acid- (SEQ ID NO: 35)WSNGCNYTNWSNYTNGGNGCNGAYGARGCNACNGARWSNATHATHCAYMG N  81-16 peptide-(SEQ ID NO: 16) SADERTESIIHR nucleic acid- (SEQ ID NO: 36)WSNGCNGAYGARMGNACNGARWSNATHATHCAYMGN 81-17 peptide- (SEQ ID NO: 17)SALSLFADERTESIIHR nucleic acid- (SEQ ID NO: 37)WSNGCNYTNWSNYTNTTYGCNGAYGARMGNACNGARWSNATHATHCAYMG N  81-18 peptide-(SEQ ID NO: 18) SALSL(L-1-NAL)ADERTESIIHR nucleic acid- (SEQ ID NO: 38)  WSNGCNYTNWSNYTN (SEQ ID NO: 39)GCNGAYGARMGNACNGARWSNATHATHCAYMGN 81-19 peptide- (SEQ ID NO: 19)FGADERTESIIHR nucleic acid- (SEQ ID NO: 40)TTYGGNGCNGAYGARMGNACNGARWSNATHATHCAYMGN 81-20 peptide-  (SEQ ID NO: 20)L-1-NALGADERTESIIHR nucleic acid- (SEQ ID NO: 41)GCNYTNGGNGCNGAYGARMGNACNGARWSNATHATHCAYMGN P78 amino acid sequence:(SEQ ID NO: 42) VLLSPLSVATALSALSLGADERTESIIHRALYYDLISSPDIHGTnucleic acid sequence: (SEQ ID NO: 43)GTGCTGCTGAGCCCGCTGTCGGTGGCAACCGCGCTGAGCGCTCTGTCACTGGGCGCAGATGAACGTACTGAATCCATTATTCATCGCGCGCTGTATTATGACCTGATTAGCTCTCCAGACATTCATGGCACC

TABLE 4 Nucleic acid code to generate computed sequences of peptide 81-1to 81-20 Comple- Oppo- Code Meaning Etymology ment site A A Adenosine TB T/U T or U Thymidine/Uridine A V G G Guanine C H C C Cytidine G D K Gor T Keto M M M A or C Amino K K R A or G Purine Y Y Y C or T PyrimidineR R S C or G Strong S W W A or T Weak W S B C or G or T not A (B comesafter A) V A V A or C or G not T/U (V comes after B T/U U) H A or C or Tnot G (H comes after G) D G D A or G or T not C (D comes after C) H CX/N G or A or T or C any N . . not G or A or T . N or C — gap ofindeterminate length

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety.

While the invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

1-36. (canceled)
 37. A composition comprising at least one peptideanalog of pigment epithelium derived factor (PEDF), wherein the at leastone peptide analog comprises at least one selected from the groupconsisting of SpxA1 (SEQ ID NO: 44), 81-1 (SEQ ID NO: 1), 81-2 (SEQ IDNO: 2), 81-6 (SEQ ID NO: 6), 81-7 (SEQ ID NO: 7), 81-8 (SEQ ID NO: 8),81-9 (SEQ ID NO: 9), 81-11 (SEQ ID NO: 11), 81-12 (SEQ ID NO: 12), 81-13(SEQ ID NO: 13), 81-14 (SEQ ID NO: 14), 81-15 (SEQ ID NO: 15), 81-16(SEQ ID NO: 16), 81-17 (SEQ ID NO: 17), 81-18 (SEQ ID NO: 18), 81-19(SEQ ID NO: 19), and 81-20 (SEQ ID NO: 20).
 38. The composition of claim37, wherein the composition further comprises a pharmaceutical carrier.39. The composition of claim 37, wherein the at least one peptide analogcomprises at least one peptide analog selected from the group consistingof SpxA1 (SEQ ID NO: 44), 81-2 (SEQ ID NO: 2), 81-12 (SEQ ID NO: 12),81-13 (SEQ ID NO: 13), and 81-20 (SEQ ID NO: 20).
 40. The composition ofclaim 37, wherein the composition is configured for delivery to the eyeof a subject.
 41. The composition of claim 37, wherein the compositionis formulated as an eye drop, a gel, a foam or an injectate.
 42. Amethod for reducing inflammation in a subject in need thereof, themethod comprising administering to a subject a therapeutically effectiveamount of a composition comprising at least one peptide analog of PEDF.43. The method of claim 42, wherein the at least one peptide analogcomprises at least one selected from the group consisting of SpxA1 (SEQID NO: 44), 81-1 (SEQ ID NO: 1), 81-2 (SEQ ID NO: 2), 81-6 (SEQ ID NO:6), 81-7 (SEQ ID NO: 7), 81-8 (SEQ ID NO: 8), 81-9 (SEQ ID NO: 9), 81-11(SEQ ID NO: 11), 81-12 (SEQ ID NO: 12), 81-13 (SEQ ID NO: 13), 81-14(SEQ ID NO: 14), 81-15 (SEQ ID NO: 15), 81-16 (SEQ ID NO: 16), 81-17(SEQ ID NO: 17), 81-18 (SEQ ID NO: 18), 81-19 (SEQ ID NO: 19), and 81-20(SEQ ID NO: 20).
 44. The method of claim 42, wherein the composition isformulated as an eye drop, a gel, a foam or an injectate.
 45. The methodof claim 42, wherein the disease or disorder is associated withangiogenesis.
 46. The method of claim 42, wherein the disease ordisorder is selected from the group consisting of diabetic retinopathy,retinopathy of prematurity, age-related macular degeneration, retinitispigmentosis, glaucoma, uveitis, corneal inflammation, diabetes, aneurodegenerative disease, nerve injury, sepsis, acute respiratorydistress syndrome, nephrotic syndrome, diabetic neuropathy,preproliferative diabetic retinopathy, proliferative diabeticretinopathy, cancer, and cystic fibrosis.
 47. The method of claim 42,wherein the subject is a mammal.
 48. The method of claim 42, wherein themammal is a human.
 49. A method of reducing the level of VEGF in asubject, the method comprising administering to a subject a compositioncomprising at least one peptide analog of PEDF.
 50. The method of claim49, wherein the at least one peptide analog comprises at least oneselected from the group consisting of SpxA1 (SEQ ID NO: 44), 81-1 (SEQID NO: 1), 81-2 (SEQ ID NO: 2), 81-6 (SEQ ID NO: 6), 81-7 (SEQ ID NO:7), 81-8 (SEQ ID NO: 8), 81-9 (SEQ ID NO: 9), 81-11 (SEQ ID NO: 11),81-12 (SEQ ID NO: 12), 81-13 (SEQ ID NO: 13), 81-14 (SEQ ID NO: 14),81-15 (SEQ ID NO: 15), 81-16 (SEQ ID NO: 16), 81-17 (SEQ ID NO: 17),81-18 (SEQ ID NO: 18), 81-19 (SEQ ID NO: 19), and 81-20 (SEQ ID NO: 20).51. A method of culturing a stem cell comprising contacting the stemcell with at least one peptide analog of PEDF, wherein the at leastpeptide analog comprises at least one selected from the group consistingof SpxA1 (SEQ ID NO: 44), 81-1 (SEQ ID NO: 1), 81-2 (SEQ ID NO: 2), 81-6(SEQ ID NO: 6), 81-7 (SEQ ID NO: 7), 81-8 (SEQ ID NO: 8), 81-9 (SEQ IDNO: 9), 81-11 (SEQ ID NO: 11), 81-12 (SEQ ID NO: 12), 81-13 (SEQ ID NO:13), 81-14 (SEQ ID NO: 14), 81-15 (SEQ ID NO: 15), 81-16 (SEQ ID NO:16), 81-17 (SEQ ID NO: 17), 81-18 (SEQ ID NO: 18), 81-19 (SEQ ID NO:19), and 81-20 (SEQ ID NO: 20).
 52. The method of claim 51, wherein themethod comprises contacting the cell with a liquid medium comprising theat least one peptide analog.
 53. The method of claim 52, wherein thecomposition is formulated as an implant wherein the implant releases thepeptide analog.